Compositions and Methods for Diagnosing and Treating Diseases and Disorders Associated With D-DT

ABSTRACT

The present invention relates to the discovery that altered levels of D-DT (also known as MIF-2) are associated with disorders and diseases. Thus, the present invention relates to compositions and methods useful of the assessment, diagnosis, characterization, prevention and treatment of disorders and diseases associated with an elevated level of D-DT. The present invention also relates to compositions and methods useful of the assessment, diagnosis, characterization, prevention and treatment of disorders and diseases associated with a reduced level of D-DT.

BACKGROUND OF THE INVENTION

Macrophage migration inhibitory factor (MIF) is the first cytokineactivity described and a key regulatory mediator that is released uponactivation of different cell types (Bloom, et al., 1966, Science153:80-82; David, 1966, Proc Natl Acad Sci USA 56:72-77; Bernhagen, etal., 2003, Nature 365:756-759). MIF increases macrophage antimicrobialresponses and it is expressed upstream of cytokines such as tumornecrosis factor (TNF)-α, IFN-γ, and IL-1β (Calandra, et al., 2003 NatRev Immunol 3:791-800). MIF activates immune cells by binding to CD74,leading to the recruitment of CD44 into a signaling complex, thestimulation of nonreceptor tyrosine kinases, and initiation of theERK1/2 MAP kinase pathway (Shi, et al., 2006 Immunity 25:595-606; Leng,et al., 2003, J Exp Med 197:1467-1476). The chemokine receptors CXCR2and CXCR4 also become activated by MIF via noncognate interactions thatare reinforced in the presence of CD74 (Bernhagen, et al., 2007, Nat Med13:587-596). Among mesenchymal cell types, MIF binding to cardiomyocyteCD74 stimulates the AMP-activated kinase (AMPK) cascade to mediateprotection from ischemic injury (Miller, et al., 2008, Nature451:578-582; Qi, et al., 2009, J Clin Invest 119: 3807-3816).

Although MIF receptor knockout mice (CD74−/−) phenocopy features MIFdeficiency (Meyer-Siegler, et al., 2006, J Immunol 177:8730-8739;Topilski, et al., 2002, J Immunol 168:1610-1617), recent observationshave led to the hypothesis that there may be a second ligand for CD74.MIF-deficient B cells, for example, are more sensitive to apoptosis thanwild-type B cells, but the magnitude of this defect is twofold morepronounced in CD74-deficient cells (Gore, et al., 2008, J Biol Chem283:2784-2792). Intravital microscopy studies also have shown a morepronounced effect of antagonism of CD74 than MIF in monocyte arrest(Bernhagen, et al., 2007, Nat Med 13:587-596). Anti-MIF antibodies,although highly effective in experimental studies, do not completelyinhibit CD74-dependent cellular activation responses (Chagnon, et al.,2005, Circ Res 96:1095-1102).

D-dopachrome tautomerase (D-DT) (also known as MIF-2) and MIF show aconserved intron-exon structure and their coding regions are highlyhomologous. The genes for MIF and D-DT are in close apposition to eachother and to two theta-class glutathione S-transferases, suggesting thatthese gene clusters arose by an ancestral duplication event. D-DT wasnamed for its ability to tautomerize the nonnaturally occurring,D-stereoisomer of dopachrome, which is a catalytic property shared withMIF. This activity has been hypothesized to be a vestigial function thatreflects MIF's ancestral origin in the invertebrate melanoticencapsulation response (Fingerle-Rowson, et al., 2009, Mol Cell Biol29:1922-1932). A crystal structure of D-DT has verified its 3Dsimilarity with MIF (Sugimoto, et al., 1999, Biochemistry 38:3268-3279).With the exception of recent studies indicating an interaction betweenthe MIF and D-DT genes in the expression of proangiogenic factors andCOX-2 in adenocarcinoma cell lines (Xin, et al., 2010, Mol Cancer Res8:1601-1609; Coleman, et al., 2008, J Immunol 181:2330-2337), there havebeen no studies of the biologic functions of D-DT.

Despite the advances made in the art for detecting and treatinganimation associated with MIF signaling through CD74, there is a need inthe art for the detection and treatment of inflammation associated withother molecules that signal through CD74. The present invention fulfillsthese needs.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that altered levels ofD-DT (also known as MIF-2) are associated with disorders and diseases.Thus, the present invention relates to compositions and methods usefulfor the assessment, diagnosis, characterization, prevention andtreatment of disorders and diseases associated with an elevated level ofD-DT. The present invention also relates to compositions and methodsuseful of the assessment, diagnosis, characterization, prevention andtreatment of disorders and diseases associated with a reduced level ofD-DT.

In one embodiment, the invention is a method of diagnosing a disease ordisorder in a subject including the steps of: determining the level ofD-DT in a biological sample from the subject, comparing the level ofD-DT in the biological sample with a comparator control, and diagnosingthe subject with a disease or disorder when the level of D-DT in thebiological sample is different than the level of D-DT of the comparatorcontrol. In one embodiment, the level of D-DT in the biological sampleis elevated when compared with the comparator control, while in anotherembodiment, the level of D-DT in the biological sample is reduced whencompared with the comparator control. In one embodiment, the level ofD-DT in the biological sample is determined by measuring the level ofD-DT mRNA, while in another embodiment, the level of the level of D-DTin the biological sample is determined by measuring the level of D-DTpolypeptide. In some embodiments, the level of D-DT in the biologicalsample is determined by measuring an enzymatic activity of D-DTpolypeptide in the biological sample. In another embodiment, the levelof D-DT in the biological sample is determined by measuring the bindingof a detectable molecule to the D-DT enzyme substrate binding site. In afurther embodiment, the level of D-DT in the biological sample isdetermined by measuring the displacement of a detectable molecule fromthe D-DT enzyme substrate binding site. In various embodiments, thecomparator control is at least one selected from the group consistingof: a positive control, a negative control, a historical control, ahistorical norm, or the level of a reference molecule in the biologicalsample. In a particular embodiment, the reference molecule is MIF. Themethod of diagnosing a disease or disorder of the present inventionuseful in diagnosing a variety of diseases and disorders associated withD-DT, including, for example, infection, inflammatory disease,autoimmunity, cancer and ischemia-reperfusion injury. In preferredembodiments, the subject is human.

In another embodiment, the invention is a composition comprising a D-DTinhibitor. In various embodiments, the D-DT inhibitor is an antibodythat specifically binds to D-DT, an antibody that specifically binds toD-DT and does not specifically bind to MIF, or an antibody thatspecifically binds to D-DT and also specifically binds to MIF. Invarious embodiments, the D-DT antibody is at least one of a polyclonalantibody, a monoclonal antibody, an intracellular antibody, an antibodyfragment, a single chain antibody (scFv), a heavy chain antibody, asynthetic antibody, a chimeric antibody, and humanized antibody. Inanother embodiment, the D-DT inhibitor is an antisense nucleic acid. Insome embodiments, the antisense nucleic acid is an siRNA or an miRNA. Ina particular embodiment, the D-DT inhibitor is an siRNA comprising thenucleic acid sequence of SEQ ID NO: 2. In other various embodiments, theD-DT inhibitor is at least one of a chemical compound, a protein, apeptide, a peptidomemetic, a ribozyme, or a small molecule chemicalcompound.

In one embodiment, the invention is a method of treating a disease ordisorder in a subject by administering to the subject a therapeuticallyeffective amount of a composition comprising a D-DT inhibitor. Invarious embodiments, the D-DT inhibitor is an antibody that specificallybinds to D-DT, an antibody that specifically binds to D-DT and does notspecifically bind to MIF, or an antibody that specifically binds to D-DTand also specifically binds to MIF. In various embodiments, the D-DTantibody is at least one of a polyclonal antibody, a monoclonalantibody, an intracellular antibody, an antibody fragment, a singlechain antibody (scFv), a heavy chain antibody, a synthetic antibody, achimeric antibody, and humanized antibody. In another embodiment, theD-DT inhibitor is an antisense nucleic acid. In some embodiments, theantisense nucleic acid is an siRNA or an miRNA. In a particularembodiment, the D-DT inhibitor is an siRNA comprising the nucleic acidsequence of SEQ ID NO: 2. In other various embodiments, the D-DTinhibitor is at least one of a chemical compound, a protein, a peptide,a peptidomemetic, a ribozyme, or a small molecule chemical compound. Invarious embodiments, the disease or disorder is at least one ofinfection, inflammatory disease, autoimmunity and cancer. In preferredembodiments, the subject is human.

In another embodiment, the invention is a method of preventing a diseaseor disorder in a subject by administering to the subject atherapeutically effective amount of composition comprising a D-DTinhibitor. In various embodiments, the D-DT inhibitor is an antibodythat specifically binds to D-DT, an antibody that specifically binds toD-DT and does not specifically bind to MIF, or an antibody thatspecifically binds to D-DT and also specifically binds to MIF. Invarious embodiments, the D-DT antibody is at least one of a polyclonalantibody, a monoclonal antibody, an intracellular antibody, an antibodyfragment, a single chain antibody (scFv), a heavy chain antibody, asynthetic antibody, a chimeric antibody, and humanized antibody. Inanother embodiment, the D-DT inhibitor is an antisense nucleic acid. Insome embodiments, the antisense nucleic acid is an siRNA or an miRNA. Ina particular embodiment, the D-DT inhibitor is an siRNA comprising thenucleic acid sequence of SEQ ID NO: 2. In other various embodiments, theD-DT inhibitor is at least one of a chemical compound, a protein, apeptide, a peptidomemetic, a ribozyme, or a small molecule chemicalcompound. In various embodiments, the disease or disorder is at leastone of infection, inflammatory disease, autoimmunity and cancer. Inpreferred embodiments, the subject is human.

In one embodiment, the invention is a composition comprising a D-DTactivator. In other various embodiments, the D-DT activator is at leastone of a chemical compound, a protein, a peptide, a peptidomemetic, anantisense nucleic acid, a ribozyme, or a small molecule chemicalcompound.

In another embodiment, the invention is a method of treatingischemia-reperfusion injury in a subject by administering to the subjecta therapeutically effective amount of a composition comprising at leastone of a D-DT polypeptide, a recombinant D-DT polypeptide, an activeD-DT polypeptide fragment, or a D-DT activator. In a preferredembodiment, the subject is a human.

In a further embodiment, the invention is a method of preventingischemia-reperfusion injury in a subject by administering to the subjecta therapeutically effective amount of a composition comprising at leastone of a D-DT polypeptide, a recombinant D-DT polypeptide, an activeD-DT polypeptide fragment, or a D-DT activator. In a preferredembodiment, the subject is a human.

In yet another embodiment, the invention is a method of preventingischemia-reperfusion injury in a tissue or organ by administering to thetissue or organ a therapeutically effective amount of a compositioncomprising at least one selected from the group consisting of: a D-DTpolypeptide, a recombinant D-DT polypeptide, an active D-DT polypeptidefragment, or a D-DT activator. In some embodiments, the tissue or organis a pre-transplant tissue or organ. In a preferred embodiment, thetissue or organ is human.

In another embodiment, the invention is a method of identifying a testcompound as a modulator of D-DT including the steps of: determining thelevel of D-DT in the presence of a test compound, determining the levelof D-DT in the absence of a test compound, comparing the level of D-DTin the presence of the test compound with the level of D-DT in theabsence of the test compound, and identifying the test compound as amodulator of D-DT when the level of D-DT in the presence of the testcompound is different than the level of D-DT in the absence of the testcompound. In some embodiments, the test compound is identified as a D-DTactivator when the level of D-DT is higher in the presence of the testcompound. In other embodiments, the test compound is identified as aD-DT inhibitor when the level of D-DT is lower in the presence of thetest compound. In some embodiments, the level of D-DT is determined bymeasuring the level of D-DT mRNA. In other embodiments, the level ofD-DT is determined by measuring the level of D-DT polypeptide. In someembodiments, the level of D-DT is determined by measuring an enzymaticactivity of D-DT polypeptide. In certain embodiments, the enzymaticactivity is tautomerase activity. In some embodiments, the level of D-DTis determined by measuring the binding of a detectable molecule to theD-DT enzyme substrate binding site. In other embodiments, the level ofD-DT is determined by measuring the displacement of a detectablemolecule from the D-DT enzyme substrate binding site. In variousembodiments, the test compound is at least one of: a chemical compound,a protein, a peptide, a peptidomemetic, an antibody, a nucleic acid, anantisense nucleic acid, a ribozyme, and a small molecule chemicalcompound.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIGS. 1A and 1B, is a schematic depicting the genomicorganization and protein homology of MIF and D-DT (also known as MIF-2).(1A) Schematic diagrams showing the relationship between human MIF andD-DT with matrix metalloprotease 11 and GST genes (Upper) and the aminoacid sequence and secondary structure homologies of the two proteins(Lower). (1B) Mouse Mif, D-DT, and adjacent genes (Upper) and thecorresponding amino acid sequence and secondary structure homologies ofthe two proteins (Lower). Gene structure was compiled fromwww.ensembl.org and sequence alignment performed using ClustalX andespript.ibcp.fr/ESPript/ESPript.

FIG. 2 depicts the results of experiments characterizing the D-DTprotein. Characterization of the D-DT protein. (A) SDS/PAGE andCoomassie analysis of sequential purification steps of recombinant D-DTprotein. The examples shown are for mouse D-DT but qualitativelyidentical results were obtained for human D-DT. (B) Electrosprayionization mass spectrometry of mouse D-DT showing a molecular mass(m/z) that is within 0.02% accuracy of the predicted m/z (12,946 Da).(C) Tautomerization activity of human MIF and D-DT measured with thesubstrate, p-hydoxyphenylpyruvate. Results are expressed as mean±SD ofduplicate measurements and are representative of three experiments. (D)Anti-D-DT antibody specifically recognizes D-DT. (Left) Anti-D-DTantibody recognizes recombinant murine D-DT protein in Western blotting(1-100 ng/lane), but does not detect recombinant mouse MIF. (Right) D-DTELISA quantifies concentrations in the picogram range and shows nocrossreactivity to MIF. Results are expressed as mean±SD of duplicatemeasurements and are representative for two independent experiments.

FIG. 3, comprising FIGS. 3A-3E, depicts the results of experimentsdemonstrating the D-DT binds with high affinity to the MIF receptor.D-DT binds with high affinity to the MIF receptor, CD74. (3A)Concentration-dependent binding of D-DT and MIF to the MIF receptorectodomain, sCD74, using biotinylated human MIF as competitor.Heat-denatured MIF served as a negative control. (3B) Real-time surfaceplasmon resonance analysis (BIAcore) of the interaction between D-DT andsCD74. (3C) Coimmunoprecipitation of D-DT/JAB1 and MIF/JAB1. (Left)Cells were lysed and recombinant D-DT was added. JAB1/D-DT-containingprotein complexes were coprecipitated by pull-down of JAB1, and D-DT wasdetected by Western blot. (Right) Coimmunoprecipitation between JAB1 andMIF following the same protocol. (3D) D-DT is differentially expressedin mouse tissue. Protein lysates (75 μg) were separated by SDS/PAGE andanalyzed by Western blot for D-DT, MIF, and CD74 (n=2 mice studied).(3E) D-DT protein expression analyzed by immunostaining of fiverepresentative organs from a C57BL/6 mouse (n=3 mice studied).

FIG. 4, comprising FIGS. 4A-4C, depicts the results of experimentsassessing the functional comparison of D-DT and MIF. (4A) D-DT activatesthe sustained ERK1/2 MAP kinase pathway in a MIF receptor complex(CD74/CD44)-dependent manner. (Top) Macrophages (1×10⁶/mL) were treatedwith 0, 10, or 50 ng/mL of D-DT or MIF for 2 hours. Cell lysates wereanalyzed for phosphorylation of ERK1/2. (Middle) Macrophages weretreated with the indicated concentrations of D-DT, MIF, or D-DT plusMIF. Lysates were analyzed for the phosphorylation status of ERK1/2.(Bottom) Wild-type and MIF receptor knockout (CD74−/− or CD44−/−)macrophages were treated with 50 ng/mL of D-DT or MIF for 2 hours, andcell lysates were analyzed by Western blot. Results are representativeof at least two independent experiments. (4B) Increasing concentrationsof D-DT or MIF inhibit the chemotaxis of human peripheral bloodmonocytes to MCP-1. Data shown are mean±SD of quadruplicate assays andstatistical significance for the comparison of MIF vs. D-DT was analyzedby an unpaired Student's t test; *P<0.01. (4C) D-DT or MIF inhibitsglucocorticoid-mediated suppression of TNF production. Macrophages werepreincubated for 1 hour with or without dexamethasone (Dex, 100 nM),MIF, or D-DT (100 ng/mL) and then stimulated with LPS (100 ng/mL).Supernatants were collected after 4 hours and TNF was quantified byELISA. Data shown are mean±SD of triplicate samples from one experimentand are representative of four independent experiments. *P<0.005,**P<0.001 vs. LPS+Dex condition by an unpaired Student's t test.

FIG. 5, comprising FIGS. 5A-5E, depicts the results of experimentsdemonstrating that the neutralization of D-DT protects from lethalendotoxic shock. (5A) D-DT is released from macrophages after LPSstimulation. Peritoneal macrophages (1×106/mL) were stimulated with LPSor PBS (control) and supernatants were analyzed by ELISA for D-DT andMIF content. Results are expressed as mean±SD of duplicate assays andare representative of at least three independent experiments. (5B)Reciprocal regulation of MIF and D-DT. Macrophages were transfected withMIF, D-DT, or control siRNA, respectively, and cultivated for 4 days.(Upper) Cells were lysed and a Western blot was performed. (Lower)Macrophages were stimulated with LPS (1 ng/mL) and after 6 hours thesupernatants were collected for ELISA. (5C) LPS challenge leads toincreased D-DT concentrations in serum. BALB/c mice (8 wk, female) werechallenged with 12.5 mg/kg of LPS, and blood was drawn 0, 6, 12, and 24hours after i.p. LPS administration. Serum was analyzed by ELISA forD-DT and MIF content. The results are expressed as mean values±SD of twoindependent experiments (n=10), and statistical significance was byStudent's t test, **P<0.01, ***P<0.001. (5D) Neutralization of D-DTprotects from lethal endotoxemia. BALB/c mice were injected i.p. withanti-D-DT antibody or nonimmune antibody (control) 2 hours before LPSadministration (20 mg/kg). Data points are from three independentexperiments. Survival was 75% (15 of 20) in mice treated with anti-D-DTantibody and 19% (4 of 21) in mice treated with control antibody.P<0.0001, Kaplan-Meier test. (5E) Neutralization of D-DT influencesserum cytokine concentrations. Mice were treated with LPS as in C andblood was drawn for cytokine analysis by Luminex. The results areexpressed as mean values±SD of three independent experiments (Student'st test, *P<0.05, **P<0.01).

FIG. 6, comprising FIGS. 6A-6D, depicts the results of experimentsdemonstrating that human serum concentrations of D-DT correlate withMIF, sepsis severity, and the presence of ovarian cancer. (6A) D-DT andMIF are elevated in the serum of patients with sepsis. Medianconcentrations of D-DT and MIF in healthy controls were 6.9 ng/mL and6.3 ng/mL, respectively. In patients with sepsis, the medianconcentrations were 56 ng/mL for D-DT and 111 ng/mL for MIF (*P<0.0001by nonparametric t test). ROC analysis revealed an area under the curveof 0.99 for both proteins. (6B) Positive correlation between the APACHEII (sepsis severity) scores and the levels of either D-DT or MIF. (6C)D-DT and MIF show a significant correlation both in the serum of healthyindividuals and in the serum of patients with severe sepsis. (6D) D-DTand MIF are elevated in the serum of patients with ovarian cancer andshow a positive correlation. Statistical significance between sera fromhealthy and diseased individuals was determined by nonparametric t test,*P<0.001, and the significance of correlation was by Pearsoncalculation.

FIG. 7 depicts the results of experiments demonstrating that human serumconcentrations of D-DT correlate with MIF and the presence ofvasculitis.

FIG. 8 depicts the results of an example western blot experimentdemonstrating that D-DT activates AMPK.

FIG. 9 depicts the results of experiments demonstrating that endogenousDDT has a role in mediating AMPK activation during hypoxia in isolatedrat heart left ventricular muscles. The addition of purified rabbitpolyclonal neutralizing antibody for 30 minutes prior to and during 15minutes of hypoxia significantly reduced the critical phosphorylation ofthreonine 172 in the activating domain of the alpha catalytic subunit ofAMP-activated protein kinase. DDT antibody also decreased thephosphorylation of downstream acetyl-CoA carboxylase. Control wasperformed with non-immune IgG incubation. FIG. 9B shows a comparison toincubation with MIF neutralizing antibody. p=0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that altered levels ofD-DT (also known as MIF-2) are associated with disorders and diseases.Thus, the present invention relates to compositions and methods usefulfor the assessment, diagnosis, characterization, prevention andtreatment of disorders and diseases associated with an elevated level ofD-DT. The present invention also relates to compositions and methodsuseful of the assessment, diagnosis, characterization, prevention andtreatment of disorders and diseases associated with a reduced level ofD-DT.

In some embodiments, the compositions of the invention relate toinhibitors of D-DT. The methods of the invention include methods ofdiagnosing disorders and diseases associated with elevated levels ofD-DT, as well as methods of monitoring the effectiveness of an appliedtreatment regimen of a disorder or disease associated with an elevatedlevel of D-DT. In various embodiments, the disorders and diseases thatcan be diagnosed, assessed, characterized, prevented or treated usingthe compositions and methods of the invention include infection,inflammatory disease, autoimmunity and cancer.

In other embodiments, the compositions of the invention relate toactivators of D-DT. The methods of the invention include methods ofdiagnosing disorders and diseases associated with reduced levels ofD-DT, as well as methods of monitoring the effectiveness of an appliedtreatment regimen of a disorder or disease associated with a reducedlevel of D-DT. In various embodiments, the disorders and diseases thatcan be diagnosed, assessed, characterized, prevented or treated usingthe compositions and methods of the invention includeischemia-reperfusion injury.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

An “allele” refers to one specific form of a genetic sequence (such as agene) within a cell, an individual or within a population, the specificform differing from other forms of the same gene in the sequence of atleast one, and frequently more than one, variant sites within thesequence of the gene. The sequences at these variant sites that differbetween different alleles are termed “variants.” “polymorphisms,” or“mutations.”

As used herein, to “alleviate” a disease means reducing the frequency orseverity of at least one sign or symptom of a disease or disorder.

As used herein the terms “alteration,” “defect,” “variation,” or“mutation,” refers to a mutation in a gene in a cell that affects thefunction, activity, expression (transcription or translation) orconformation of the polypeptide that it encodes. Mutations encompassedby the present invention can be any mutation of a gene in a cell thatresults in the enhancement or disruption of the function, activity,expression or conformation of the encoded polypeptide, including thecomplete absence of expression of the encoded protein and can include,for example, missense and nonsense mutations, insertions, deletions,frameshifts and premature terminations. Without being so limited,mutations encompassed by the present invention may alter splicing themRNA (splice site mutation) or cause a shift in the reading frame(frameshift).

The term “amplification” refers to the operation by which the number ofcopies of a target nucleotide sequence present in a sample ismultiplied.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies(scFv), heavy chain antibodies, such as camelid antibodies, syntheticantibodies, chimeric antibodies, and humanized antibodies (Harlow etal., 1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

As used herein, an “immunoassay” refers to any binding assay that usesan antibody capable of binding specifically to a target molecule todetect and quantify the target molecule.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,an iontophoresis device, a patch, and the like, for administering thecompositions of the invention to a subject.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “cancer,” or “neoplasm” as used herein includes, but is notlimited to, benign and malignant cancers of the oral cavity (e.g.,mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus,stomach, small intestine, colon, rectum, liver, bile duct, gall bladder,pancreas, etc.), respiratory system (e.g., larynx, lung, bronchus,etc.), bones, joints, skin (e.g., basal cell, squamous cell, melanoma,etc.), breast, genital system, (e.g., uterus, ovary, prostate, testis,etc.), urinary system (e.g, bladder, kidney, ureter, etc.), eye, nervoussystem (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), andhematopoietic system (e.g., lymphoma, myeloma, leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, acute myeloidleukemia, chronic myeloid leukemia, etc.).

The term “coding sequence,” as used herein, means a sequence of anucleic acid or its complement, or a part thereof, that can betranscribed and/or translated to produce the mRNA and/or the polypeptideor a fragment thereof. Coding sequences include exons in a genomic DNAor immature primary RNA transcripts, which are joined together by thecell's biochemical machinery to provide a mature mRNA. The anti-sensestrand is the complement of such a nucleic acid, and the coding sequencecan be deduced therefrom. In contrast, the term “non-coding sequence,”as used herein, means a sequence of a nucleic acid or its complement, ora part thereof, that is not translated into amino acid in vivo, or wheretRNA does not interact to place or attempt to place an amino acid.Non-coding sequences include both intron sequences in genomic DNA orimmature primary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods that depend upon binding between nucleic acids.

As used herein, the term “diagnosis” refers to the determination of thepresence of a disease or disorder. In some embodiments of the presentinvention, methods for making a diagnosis are provided which permitdetermination of a the presence of a particular disease or disorder.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides; at least about 1000 nucleotides to about 1500nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about2500 nucleotides (and any integer value in between). As used herein, theterm “fragment,” as applied to a protein or peptide, refers to asubsequence of a larger protein or peptide. A “fragment” of a protein orpeptide can be at least about 20 amino acids in length; for example, atleast about 50 amino acids in length; at least about 100 amino acids inlength; at least about 200 amino acids in length; at least about 300amino acids in length; or at least about 400 amino acids in length (andany integer value in between).

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatincludes coding sequences necessary for the production of a polypeptide,precursor, or RNA (e.g., mRNA). The polypeptide may be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional property (e.g., enzymaticactivity, ligand binding, signal transduction, immunogenicity, etc.) ofthe full-length or fragment is retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends for a distance of about 2kb or more on either end such that the gene corresponds to the length ofthe full-length mRNA and 5′ regulatory sequences which influence thetranscriptional properties of the gene. Sequences located 5′ of thecoding region and present on the mRNA are referred to as 5′-untranslatedsequences. The 5′-untranslated sequences usually contain the regulatorysequences. Sequences located 3′ or downstream of the coding region andpresent on the mRNA are referred to as 3′-untranslated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene that aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

A “genome” is all the genetic material of an organism. In someinstances, the term genome may refer to the chromosomal DNA. Genome maybe multichromosomal such that the DNA is cellularly distributed among aplurality of individual chromosomes. For example, in human there are 22pairs of chromosomes plus a gender associated XX or XY pair. DNA derivedfrom the genetic material in the chromosomes of a particular organism isgenomic DNA. The term genome may also refer to genetic materials fromorganisms that do not have chromosomal structure. In addition, the termgenome may refer to mitochondria DNA. A genomic library is a collectionof DNA fragments representing the whole or a portion of a genome.Frequently, a genomic library is a collection of clones made from a setof randomly generated, sometimes overlapping DNA fragments representingthe entire genome or a portion of the genome of an organism.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “housekeeping gene” as used herein refers to genes that aregenerally always expressed and thought to be involved in routinecellular metabolism. Housekeeping genes are well known and include suchgenes as glyceraldehyde-3-phosphate dehydrogenase (G3PDH or GAPDH),albumin, actins, tubulins, cyclophilin, hypoxanthinephsophoribosyltransferase (HRPT), 28S, and 18S rRNAs and the like.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.” Asingle DNA molecule with internal complementarity could assume a varietyof secondary structures including loops, kinks or, for long stretches ofbase pairs, coils.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, and/or compound of the invention in the kit for identifying,diagnosing or alleviating or treating the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of identifying, diagnosing oralleviating the diseases or disorders in a cell or a tissue of asubject. The instructional material of the kit may, for example, beaffixed to a container that contains the nucleic acid, peptide, and/orcompound of the invention or be shipped together with a container thatcontains the nucleic acid, peptide, and/or compound. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the recipient uses the instructional material and thecompound cooperatively.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

The term “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to a probe togenerate a “labeled” probe. The label may be detectable by itself (e.g.radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable (e.g., avidin-biotin). Insome instances, primers can be labeled to detect a PCR product.

The terms “microarray” and “array” refers broadly to “DNA microarrays,”“DNA chip(s),” “protein microarrays” and “protein chip(s)” andencompasses all art-recognized solid supports, and all art-recognizedmethods for affixing nucleic acid, peptide, and polypeptide moleculesthereto. Preferred arrays typically comprise a plurality of differentnucleic acid or peptide probes that are coupled to a surface of asubstrate in different, known locations. These arrays, also described as“microarrays” or colloquially “chips” have been generally described inthe art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305,5,677,195, 5,800,992, 6,040,193, 5,424,186 and Fodor et al., 1991,Science, 251:767-777, each of which is incorporated by reference in itsentirety for all purposes. Arrays may generally be produced using avariety of techniques, such as mechanical synthesis methods or lightdirected synthesis methods that incorporate a combination ofphotolithographic methods and solid phase synthesis methods. Techniquesfor the synthesis of these arrays using mechanical synthesis methods aredescribed in, e.g., U.S. Pat. Nos. 5,384,261, and 6,040,193, which areincorporated herein by reference in their entirety for all purposes.Although a planar array surface is preferred, the array may befabricated on a surface of virtually any shape or even a multiplicity ofsurfaces. Arrays may be nucleic acids on beads, gels, polymericsurfaces, fibers such as fiber optics, glass or any other appropriatesubstrate. (See U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153,6,040,193 and 5,800,992, which are hereby incorporated by reference intheir entirety for all purposes.) Arrays may be packaged in such amanner as to allow for diagnostic use or can be an all-inclusive device;e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated in theirentirety by reference for all purposes. Arrays are commerciallyavailable from, for example, Affymetrix (Santa Clara, Calif.) andApplied Biosystems (Foster City, Calif.), and are directed to a varietyof purposes, including genotyping, diagnostics, mutation analysis,marker expression, and gene expression monitoring for a variety ofeukaryotic and prokaryotic organisms. The number of probes on a solidsupport may be varied by changing the size of the individual features.In one embodiment the feature size is 20 by 25 microns square, in otherembodiments features may be, for example, 8 by 8, 5 by 5 or 3 by 3microns square, resulting in about 2,600,000, 6,600,000 or 18,000,000individual probe features.

Assays for amplification of the known sequence are also disclosed. Forexample primers for PCR may be designed to amplify regions of thesequence. For RNA, a first reverse transcriptase step may be used togenerate double stranded DNA from the single stranded RNA. The array maybe designed to detect sequences from an entire genome; or one or moreregions of a genome, for example, selected regions of a genome such asthose coding for a protein or RNA of interest; or a conserved regionfrom multiple genomes; or multiple genomes, arrays and methods ofgenetic analysis using arrays is described in Cutler, et al., 2001,Genome Res. 11(11): 1913-1925 and Warrington, et al., 2002, Hum Mutat19:402-409 and in US Patent Pub No 20030124539, each of which isincorporated herein by reference in its entirety.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a mRNA, polypeptide, ora response in a subject compared with the level of a mRNA, polypeptideor a response in the subject in the absence of a treatment or compound,and/or compared with the level of a mRNA, polypeptide, or a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

A “mutation,” as used herein, refers to a change in nucleic acid orpolypeptide sequence relative to a reference sequence (which ispreferably a naturally-occurring normal or “wild-type” sequence), andincludes translocations, deletions, insertions, and substitutions/pointmutations. A “mutant” as used herein, refers to either a nucleic acid orprotein comprising a mutation.

A “nucleic acid” refers to a polynucleotide and includespoly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acidsaccording to the present invention may include any polymer or oligomerof pyrimidine and purine bases, preferably cytosine, thymine, anduracil, and adenine and guanine, respectively. (See Albert L. Lehninger,Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is hereinincorporated in its entirety for all purposes). Indeed, the presentinvention contemplates any deoxyribonucleotide, ribonucleotide orpeptide nucleic acid component, and any chemical variants thereof, suchas methylated, hydroxymethylated or glucosylated forms of these bases,and the like. The polymers or oligomers may be heterogeneous orhomogeneous in composition, and may be isolated from naturally occurringsources or may be artificially or synthetically produced. In addition,the nucleic acids may be DNA or RNA, or a mixture thereof, and may existpermanently or transitionally in single-stranded or double-strandedform, including homoduplex, heteroduplex, and hybrid states.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 25 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides includesequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ormimetics thereof which may be isolated from natural sources,recombinantly produced or artificially synthesized. A further example ofa polynucleotide of the present invention may be a peptide nucleic acid(PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated byreference in its entirety.) The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this disclosure. It willbe understood that when a nucleotide sequence is represented herein by aDNA sequence (e.g., A, T, G, and C), this also includes thecorresponding RNA sequence (e.g., A, U, G, C) in which “U” replaces “T”.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis (U.S. Pat. Nos. 4,683,195 4,683,202, and4,965,188, hereby incorporated by reference), which describe a methodfor increasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”. As used herein, the terms “PCR product,” “PCRfragment,” “amplification product” or “amplicon” refer to the resultantmixture of compounds after two or more cycles of the PCR steps ofdenaturation, annealing and extension are complete. These termsencompass the case where there has been amplification of one or moresegments of one or more target sequences.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences.

The term “perfect match,” “match,” “perfect match probe” or “perfectmatch control” refers to a nucleic acid that has a sequence that isperfectly complementary to a particular target sequence. The nucleicacid is typically perfectly complementary to a portion (subsequence) ofthe target sequence. A perfect match (PM) probe can be a “test probe,” a“normalization control” probe, an expression level control probe and thelike. A perfect match control or perfect match is, however,distinguished from a “mismatch” or “mismatch probe.” The term“mismatch,” “mismatch control” or “mismatch probe” refers to a nucleicacid whose sequence is not perfectly complementary to a particulartarget sequence. As a non-limiting example, for each mismatch (MM)control in a high-density probe array there typically exists acorresponding perfect match (PM) probe that is perfectly complementaryto the same particular target sequence. The mismatch may comprise one ormore bases. While the mismatch(es) may be located anywhere in themismatch probe, terminal mismatches are less desirable because aterminal mismatch is less likely to prevent hybridization of the targetsequence. In a particularly preferred embodiment, the mismatch islocated at or near the center of the probe such that the mismatch ismost likely to destabilize the duplex with the target sequence under thetest hybridization conditions.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to contain non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, contemplated are alterationsof a wild type or synthetic gene, including but not limited to deletion,insertion, substitution of one or more nucleotides, or fusion to otherpolynucleotide sequences.

The term “primer” refers to an oligonucleotide capable of acting as apoint of initiation of synthesis along a complementary strand whenconditions are suitable for synthesis of a primer extension product. Thesynthesizing conditions include the presence of four differentdeoxyribonucleotide triphosphates and at least onepolymerization-inducing agent such as reverse transcriptase or DNApolymerase. These are present in a suitable buffer, which may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. A primer is preferably asingle strand sequence, such that amplification efficiency is optimized,but double stranded sequences can be utilized.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

The term “reaction mixture” or “PCR reaction mixture” or “master mix” or“master mixture” refers to an aqueous solution of constituents in a PCRreaction that can be constant across different reactions. An exemplaryPCR reaction mixture includes buffer, a mixture of deoxyribonucleosidetriphosphates, primers, probes, and DNA polymerase. Generally, templateRNA or DNA is the variable in a PCR.

“Sample” or “biological sample” as used herein means a biologicalmaterial isolated from a subject. The biological sample may contain anybiological material suitable for detecting a mRNA, polypeptide or othermarker of a physiologic or pathologic process in a subject, and maycomprise fluid, tissue, cellular and/or non-cellular material obtainedfrom the individual.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under “medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely related sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

As used herein, “substantially purified” refers to being essentiallyfree of other components. For example, a substantially purified cell isa cell which has been separated from other cell types with which it isnormally associated in its naturally occurring state. In some instances,a population of substantially purified cells refers to a homogenouspopulation of cells. In other instances, this term refers simply to acell that have been separated from the cells with which they arenaturally associated in their natural state.

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Targets are sometimes referred to in the art asanti-probes. As the term targets is used herein, no difference inmeaning is intended.

As used herein, the terms “therapy” or “therapeutic regimen” refer tothose activities taken to alleviate or alter a disorder or diseasestate, e.g., a course of treatment intended to reduce or eliminate atleast one sign or symptom of a disease or disorder usingpharmacological, surgical, dietary and/or other techniques. Atherapeutic regimen may include a prescribed dosage of one or more drugsor surgery. Therapies will most often be beneficial and reduce oreliminate at least one sign or symptom of the disorder or disease state,but in some instances the effect of a therapy will have non-desirable orside-effects. The effect of therapy will also be impacted by thephysiological state of the subject, e.g., age, gender, genetics, weight,other disease conditions, etc.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

As used herein, the term “wild-type” refers to a gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” refers to a gene or geneproduct that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics (including altered nucleic acid sequences) whencompared to the wild-type gene or gene product.

DESCRIPTION

The present invention relates to the discovery that altered levels ofD-DT (also known as MIF-2) are associated with disorders and diseases.Thus, the present invention relates to compositions and methods usefulfor the assessment, diagnosis, characterization, prevention andtreatment of disorders and diseases associated with an elevated level ofD-DT. The present invention also relates to compositions and methodsuseful of the assessment, diagnosis, characterization, prevention andtreatment of disorders and diseases associated with a reduced level ofD-DT.

In some embodiments, the compositions of the invention relate toinhibitors of D-DT. The methods of the invention include methods ofdiagnosing disorders and diseases associated with elevated levels ofD-DT, as well as methods of monitoring the effectiveness of an appliedtreatment regimen of a disorder or disease associated with an elevatedlevel of D-DT. In various embodiments, the disorders and diseases thatcan be diagnosed, assessed, characterized, prevented or treated usingthe compositions and methods of the invention include infection,inflammatory disease, autoimmunity and cancer. By way of non-limitingexamples, the disorders and diseases that can be diagnosed, assessed,characterized, prevented or treated using the compositions and methodsof the invention include: asthma, atheroma, atherosclerosis, autism,autoinflammatory disease, autoimmune myocarditis, autoimmune hepatitis,bacterial infection, cancer, celiac disease, cellular proliferativedisorder, Crohn's disease, colitis, diabetes, dermatitis,diverticulitis, dry age-related macular degeneration (AMD), endotoxemia,glomerulonephritis, graft versus host disease, Guillain-Barre syndrome,heart disease, hepatitis, inflammation, inflammatory breast cancer,inflammatory demyelinating polyneuropathy, intestinal cystitis,irritable bowel disease (IBD), lupus erythematous, microbial infection,multiple sclerosis, neoplasia, ovarian cancer, pelvic inflammatorydisease (PID), prostatitis, psoriasis, reperfusion injury, rheumatoidarthritis, sarcoidosis, sepsis, septic shock, transplant rejection,trauma-induced inflammation, ulcerative colitis, vasculitis, Wegener'sgranulomatous and wet age-related macular degeneration (AMD).

The present invention also relates to the discovery that signaling byD-DT through CD74 activates AMPK in cardiomyocytes. Thus, in otherembodiments, the compositions of the invention relate to activators ofD-DT. The methods of the invention include methods of diagnosingdisorders and diseases associated with reduced levels of D-DT, as wellas methods of monitoring the effectiveness of an applied treatmentregimen of a disorder or disease associated with a reduced level ofD-DT. In various embodiments, the disorders and diseases that can bediagnosed, assessed, characterized, prevented or treated using thecompositions and methods of the invention include ischemia-reperfusioninjury in, for example, the heart and other solid organs, including, butnot limited to, the kidney, the liver and the brain.

Assays

The present invention relates to the discovery that altered levels ofD-DT are associated with infection, inflammatory disease, autoimmunity,cancer and ischemia-reperfusion injury. In some embodiments, theinvention relates to a screening assay of a subject to determine whetherthe subject has an elevated level of D-DT. In other embodiments, theinvention relates to a screening assay of a subject to determine whetherthe subject has a reduced level of D-DT. The present invention providesmethods of assessing the level of D-DT in a subject. In variousembodiments, the level of D-DT in the biological sample can bedetermined by assessing the amount of D-DT polypeptide present in thebiological sample, the amount of D-DT mRNA present in the biologicalsample, the amount of D-DT enzymatic activity in the biological sample,the amount of D-DT receptor binding activity in the biological sample,or a combination thereof.

The present invention also provides methods of diagnosing a subjecthaving an elevated level of D-DT, with a disease or disorder, such asinfection, inflammatory disease, autoimmunity and cancer. Further, thepresent invention provides methods of diagnosing a subject having areduced level of D-DT, with a disease or disorder, such asischemia-reperfusion injury.

In one embodiment, the method of the invention is a diagnostic assay fordiagnosing infection, inflammatory disease, autoimmunity or cancer in asubject in need thereof, by determining whether the level of D-DT isincreased in a biological sample obtained from the subject. In variousembodiments, to determine whether the level of D-DT is increased in abiological sample obtained from the subject, the level of D-DT iscompared with the level of at least one comparator control, such as apositive control, a negative control, a historical control, a historicalnorm, or the level of another reference molecule in the biologicalsample. In one embodiment, the reference molecule in the biologicalsample is MIF. In certain embodiments, the ratio of D-DT and MIF isdetermined to aid in the diagnosis. The results of the diagnostic assaycan be used alone, or in combination with other information from thesubject, or other information from the biological sample obtained fromthe subject.

In another embodiment, the method of the invention is a diagnostic assayfor diagnosing ischemia-reperfusion injury in a subject in need thereof,by determining whether the level of D-DT is reduced in a biologicalsample obtained from the subject. In various embodiments, to determinewhether the level of D-DT is reduced in a biological sample obtainedfrom the subject, the level of D-DT is compared with the level of atleast one comparator control, such as a positive control, a negativecontrol, a historical control, a historical norm, or the level ofanother reference molecule in the biological sample. In one embodiment,the reference molecule in the biological sample is MIF. In certainembodiments, the ratio of D-DT and MIF is determined to aid in thediagnosis. The results of the diagnostic assay can be used alone, or incombination with other information from the subject, or otherinformation from the biological sample obtained from the subject.

In a further embodiment, the method of the invention is an assay formonitoring the effectiveness of a treatment administered to a subject inneed thereof, by determining whether the level of D-DT in a biologicalsample obtained from the subject is modulated upon administration of thetreatment. The assay can be performed before, during or after atreatment has been administered, or any combination thereof. In variousembodiments, to determine whether the level of D-DT is modulated in abiological sample obtained from the subject, the level of D-DT iscompared with the level of at least one comparator control, such as apositive control, a negative control, a historical control, a historicalnorm, or the level of another reference molecule in the biologicalsample. In one embodiment, the reference molecule in the biologicalsample is MIF. In certain embodiments, the ratio of D-DT and MIF isdetermined to aid in the monitoring of the treatment. The results of theassay can be used alone, or in combination with other information fromthe subject, or other information from the biological sample obtainedfrom the subject.

In various embodiments of the assays of the invention, the level of D-DTis determined to be elevated when the level of D-DT is increased by atleast 10%, by at least 20%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 80%, by atleast 90%, by at least 100%, by at least 125%, by at least 150%, by atleast 175%, by at least 200%, by at least 250%, by at least 300%, by atleast 400%, by at least 500%, by at least 600%, by at least 700%, by atleast 800%, by at least 900%, by at least 1000%, by at least 1500%, byat least 2000%, by at least 2500%, by at least 3000%, by at least 4000%,or by at least 5000%, when compared with a comparator control.

In other various embodiments of the assays of the invention, the levelof D-DT is determined to be reduced when the level of D-DT is reduced byat least 10%, by at least 20%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 80%, by atleast 90%, by at least 100%, by at least 125%, by at least 150%, by atleast 175%, by at least 200%, by at least 250%, by at least 300%, by atleast 400%, by at least 500%, by at least 600%, by at least 700%, by atleast 800%, by at least 900%, by at least 1000%, by at least 1500%, byat least 2000%, by at least 2500%, by at least 3000%, by at least 4000%,or by at least 5000%, when compared with a comparator control.

In the assay methods of the invention, a test biological sample from asubject is assessed for the level of D-DT in the biological sampleobtained from the patient. The level of D-DT in the biological samplecan be determined by assessing the amount of D-DT polypeptide in thebiological sample, the amount of D-DT mRNA in the biological sample, theamount of D-DT enzymatic activity in the biological sample, the amountof D-DT receptor binding activity in the biological sample, or acombination thereof. In one embodiment, the D-DT enzymatic activityassessed is D-DT tautomerization activity. In another embodiment, theD-DT enzymatic activity assessed is D-DT tautomerization ofp-hydoxyphenylpyruvate (HPP). In various embodiments, the subject is ahuman subject, and may be of any race, sex and age. Representativesubjects include those who are suspected of having infection,inflammatory disease, autoimmunity or cancer, those who have beendiagnosed with infection, inflammatory disease, autoimmunity or cancer,those who have infection, inflammatory disease, autoimmunity or cancer,those who have had infection, inflammatory disease, autoimmunity orcancer, those who at risk of a recurrence of infection, inflammatorydisease, autoimmunity or cancer, and those who are at risk of developinginfection, inflammatory disease, autoimmunity or cancer.

In various embodiments, the test sample is a sample containing at leasta fragment of a D-DT polypeptide or a D-DT nucleic acid. The term,“fragment,” as used herein, indicates that the portion of thepolypeptide, mRNA or cDNA is of a length that is sufficient to identifythe fragment as D-DT.

The test sample is prepared from a biological sample obtained from thesubject. The biological sample can be a sample from any source whichcontains a polypeptide or a nucleic acid, such as a body fluid or atissue, or a combination thereof. A biological sample can be obtained byappropriate methods, such as, by way of examples, blood draw, fluiddraw, or biopsy. A biological sample can be used as the test sample;alternatively, a biological sample can be processed to enhance access tothe polypeptides or nucleic acids, or copies of the nucleic acids, andthe processed biological sample can then be used as the test sample. Forexample, in various embodiments, nucleic acid (e.g., mRNA, cDNA preparedfrom mRNA, etc.) is prepared from a biological sample, for use in themethods. Alternatively or in addition, if desired, an amplificationmethod can be used to amplify nucleic acids comprising all or a fragmentof a mRNA in a biological sample, for use as the test sample in theassessment of the level of D-DT in the biological sample.

In various embodiments of the invention, methods of measuring D-DTpolypeptide levels in a biological sample obtained from a patientinclude, but are not limited to, an immunochromatography assay, animmunodot assay, a Luminex assay, an ELISA assay, an ELISPOT assay, aprotein microarray assay, a ligand-receptor binding assay, displacementof a ligand from a receptor assay, displacement of a ligand from ashared receptor assay, an immunostaining assay, a Western blot assay, amass spectrophotometry assay, a radioimmunoassay (RIA), aradioimmunodiffusion assay, a liquid chromatography-tandem massspectrometry assay, an ouchterlony immunodiffusion assay, reverse phaseprotein microarray, a rocket immunoelectrophoresis assay, animmunohistostaining assay, an immunoprecipitation assay, a complementfixation assay, FACS, an enzyme-substrate binding assay, an enzymaticassay, an enzymatic assay employing a detectable molecule, such as achromophore, fluorophore, or radioactive substrate, a substrate bindingassay employing such a substrate, a substrate displacement assayemploying such a substrate, and a protein chip assay (see also, 2007,Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press;2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996,Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos,Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan andRighetti, Proteomics Today, John Wiley and Sons; 2007).

In some embodiments, quantitative hybridization methods, such asSouthern analysis, Northern analysis, or in situ hybridizations, can beused (see Current Protocols in Molecular Biology, Ausubel, F. et al.,eds., John Wiley & Sons, including all supplements). A “nucleic acidprobe,” as used herein, can be a DNA probe or an RNA probe. The probecan be, for example, a gene, a gene fragment (e.g., one or more exons),a vector comprising the gene, a probe or primer, etc. For representativeexamples of use of nucleic acid probes, see, for example, U.S. Pat. Nos.5,288,611 and 4,851,330. The nucleic acid probe can be, for example, afull-length nucleic acid molecule, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to appropriate target mRNA or cDNA. The hybridization sampleis maintained under conditions which are sufficient to allow specifichybridization of the nucleic acid probe to mRNA or cDNA. Specifichybridization can be performed under high stringency conditions ormoderate stringency conditions, as appropriate. In a preferredembodiment, the hybridization conditions for specific hybridization arehigh stringency. Specific hybridization, if present, is then detectedusing standard methods. If specific hybridization occurs between thenucleic acid probe having a mRNA or cDNA in the test sample, the levelof the mRNA or cDNA in the sample can be assessed. More than one nucleicacid probe can also be used concurrently in this method. Specifichybridization of any one of the nucleic acid probes is indicative of thepresence of the mRNA or cDNA of interest, as described herein.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the quantitative hybridization methods describedherein. PNA is a DNA mimic having a peptide-like, inorganic backbone,such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, Tor U) attached to the glycine nitrogen via a methylene carbonyl linker(see, for example, 1994, Nielsen et al., Bioconjugate Chemistry 5:1).The PNA probe can be designed to specifically hybridize to a targetnucleic acid sequence. Hybridization of the PNA probe to a nucleic acidsequence is used to determine the level of the target nucleic acid inthe biological sample.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target nucleic acid sequences in the biological sampleobtained from a subject can be used to determine the level of D-DT inthe biological sample obtained from a subject. The array ofoligonucleotide probes can be used to determine the level of D-DT alone,or the level of D-DT in relation to the level of one or more othernucleic acids in the biological sample. Oligonucleotide arrays typicallycomprise a plurality of different oligonucleotide probes that arecoupled to a surface of a substrate in different known locations. Theseoligonucleotide arrays, also known as “Genechips,” have been generallydescribed in the art, for example, U.S. Pat. No. 5,143,854 and PCTpatent publication Nos. WO 90/15070 and 92/10092. These arrays cangenerally be produced using mechanical synthesis methods or lightdirected synthesis methods which incorporate a combination ofphotolithographic methods and solid phase oligonucleotide synthesismethods. See Fodor et al., Science, 251:767-777 (1991), Pirrung et al.,U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) andFodor et al., PCT Publication No. WO 92/10092 and U.S. Pat. No.5,424,186. Techniques for the synthesis of these arrays using mechanicalsynthesis methods are described in, e.g., U.S. Pat. No. 5,384,261.

After an oligonucleotide array is prepared, a nucleic acid of interestis hybridized with the array and its level is quantified. Hybridizationand quantification are generally carried out by methods described hereinand also in, e.g., published PCT Application Nos. WO 92/10092 and WO95/11995, and U.S. Pat. No. 5,424,186. In brief, a target nucleic acidsequence is amplified by well-known amplification techniques, e.g., PCR.Typically, this involves the use of primer sequences that arecomplementary to the target nucleic acid. Asymmetric PCR techniques mayalso be used. Amplified target, generally incorporating a label, is thenhybridized with the array under appropriate conditions. Upon completionof hybridization and washing of the array, the array is scanned todetermine the quantity of hybridized nucleic acid. The hybridizationdata obtained from the scan is typically in the form of fluorescenceintensities as a function of quantity, or relative quantity, of thetarget nucleic acid in the biological sample. The target nucleic acidcan be hybridized to the array in combination with one or morecomparator controls (e.g., positive control, negative control, quantitycontrol, etc.) to improve quantification of the target nucleic acid inthe sample.

The probes and primers according to the invention can be labeleddirectly or indirectly with a radioactive or nonradioactive compound, bymethods well known to those skilled in the art, in order to obtain adetectable and/or quantifiable signal; the labeling of the primers or ofthe probes according to the invention is carried out with radioactiveelements or with nonradioactive molecules. Among the radioactiveisotopes used, mention may be made of 32P, 33P, 35S or 3H. Thenonradioactive entities are selected from ligands such as biotin,avidin, streptavidin or digoxigenin, haptenes, dyes, and luminescentagents such as radioluminescent, chemoluminescent, bioluminescent,fluorescent or phosphorescent agents.

Nucleic acids can be obtained from the cells using known techniques.Nucleic acid herein refers to RNA, including mRNA, and DNA, includingcDNA. The nucleic acid can be double-stranded or single-stranded (i.e.,a sense or an antisense single strand) and can be complementary to anucleic acid encoding a polypeptide. The nucleic acid content may alsobe an RNA or DNA extraction performed on a biological sample, includinga biological fluid and fresh or fixed tissue sample.

There are many methods known in the art for the detection andquantification of specific nucleic acid sequences and new methods arecontinually reported. A great majority of the known specific nucleicacid detection and quantification methods utilize nucleic acid probes inspecific hybridization reactions. Preferably, the detection ofhybridization to the duplex form is a Southern blot technique. In theSouthern blot technique, a nucleic acid sample is separated in anagarose gel based on size (molecular weight) and affixed to a membrane,denatured, and exposed to (admixed with) the labeled nucleic acid probeunder hybridizing conditions. If the labeled nucleic acid probe forms ahybrid with the nucleic acid on the blot, the label is bound to themembrane.

In the Southern blot, the nucleic acid probe is preferably labeled witha tag. That tag can be a radioactive isotope, a fluorescent dye or theother well-known materials. Another type of process for the specificdetection of nucleic acids in a biological sample known in the art arethe hybridization methods as exemplified by U.S. Pat. No. 6,159,693 andU.S. Pat. No. 6,270,974, and related patents. To briefly summarize oneof those methods, a nucleic acid probe of at least 10 nucleotides,preferably at least 15 nucleotides, more preferably at least 25nucleotides, having a sequence complementary to a nucleic acid ofinterest is hybridized in a sample, subjected to depolymerizingconditions, and the sample is treated with an ATP/luciferase system,which will luminesce if the nucleic sequence is present. In quantitativeSouthern blotting, the level of the nucleic acid of interest can becompared with the level of a second nucleic acid of interest, and/or toone or more comparator control nucleic acids (e.g., positive control,negative control, quantity control, etc.).

Many methods useful for the detection and quantification of nucleic acidtakes advantage of the polymerase chain reaction (PCR). The PCR processis well known in the art (U.S. Pat. No. 4,683,195, U.S. Pat. No.4,683,202, and U.S. Pat. No. 4,800,159). To briefly summarize PCR,nucleic acid primers, complementary to opposite strands of a nucleicacid amplification target sequence, are permitted to anneal to thedenatured sample. A DNA polymerase (typically heat stable) extends theDNA duplex from the hybridized primer. The process is repeated toamplify the nucleic acid target. If the nucleic acid primers do nothybridize to the sample, then there is no corresponding amplified PCRproduct. In this case, the PCR primer acts as a hybridization probe.

In PCR, the nucleic acid probe can be labeled with a tag as discussedelsewhere herein. Most preferably the detection of the duplex is doneusing at least one primer directed to the nucleic acid of interest. Inyet another embodiment of PCR, the detection of the hybridized duplexcomprises electrophoretic gel separation followed by dye-basedvisualization.

Typical hybridization and washing stringency conditions depend in parton the size (i.e., number of nucleotides in length) of theoligonucleotide probe, the base composition and monovalent and divalentcation concentrations (Ausubel et al., 1994, eds Current Protocols inMolecular Biology).

In a preferred embodiment, the process for determining the quantitativeand qualitative profile of the nucleic acid of interest according to thepresent invention is characterized in that the amplifications arereal-time amplifications performed using a labeled probe, preferably alabeled hydrolysis-probe, capable of specifically hybridizing instringent conditions with a segment of the nucleic acid of interest. Thelabeled probe is capable of emitting a detectable signal every time eachamplification cycle occurs, allowing the signal obtained for each cycleto be measured.

The real-time amplification, such as real-time PCR, is well known in theart, and the various known techniques will be employed in the best wayfor the implementation of the present process. These techniques areperformed using various categories of probes, such as hydrolysis probes,hybridization adjacent probes, or molecular beacons. The techniquesemploying hydrolysis probes or molecular beacons are based on the use ofa fluorescence quencher/reporter system, and the hybridization adjacentprobes are based on the use of fluorescence acceptor/donor molecules.

Hydrolysis probes with a fluorescence quencher/reporter system areavailable in the market, and are for example commercialized by theApplied Biosystems group (USA). Many fluorescent dyes may be employed,such as FAM dyes (6-carboxy-fluorescein), or any other dyephosphoramidite reagents.

Among the stringent conditions applied for any one of thehydrolysis-probes of the present invention is the Tm, which is in therange of about 65° C. to 75° C. Preferably, the Tm for any one of thehydrolysis-probes of the present invention is in the range of about 67°C. to about 70° C. Most preferably, the Tm applied for any one of thehydrolysis-probes of the present invention is about 67° C.

In one aspect, the invention includes a primer that is complementary toa nucleic acid of interest, and more particularly the primer includes 12or more contiguous nucleotides substantially complementary to thenucleic acid of interest. Preferably, a primer featured in the inventionincludes a nucleotide sequence sufficiently complementary to hybridizeto a nucleic acid sequence of about 12 to 25 nucleotides. Morepreferably, the primer differs by no more than 1, 2, or 3 nucleotidesfrom the target flanking nucleotide sequence In another aspect, thelength of the primer can vary in length, preferably about 15 to 28nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,or 27 nucleotides in length).

Kits

The present invention also pertains to kits useful in the methods of theinvention. Such kits comprise various combinations of components usefulin any of the methods described elsewhere herein, including for example,hybridization probes or primers (e.g., labeled probes or primers),antibodies, reagents for detection of labeled molecules, materials forthe amplification of a subject's nucleic acids, a D-DT activator, a D-DTinhibitor, materials for quantitatively analyzing D-DT polypeptide orD-DT nucleic acid, materials for assessing the activity of a D-DTpolypeptide or a D-DT nucleic acid, and instructional material. Forexample, in one embodiment, the kit comprises components useful for thequantification of D-DT nucleic acid in a biological sample obtained froma subject. In another embodiment, the kit comprises components usefulfor the quantification of D-DT polypeptide in a biological sampleobtained from a subject. In a further embodiment, the kit comprisescomponents useful for the assessment of the activity (e.g., enzymaticactivity, receptor binding activity, etc.) of a D-DT polypeptide in abiological sample obtained from a subject.

In one embodiment, the kit comprises the components of a diagnosticassay for diagnosing infection, inflammatory disease, autoimmunity orcancer in a subject in need thereof, containing instructional materialand the components for determining whether the level of D-DT isincreased in a biological sample obtained from the subject. In variousembodiments, determining whether the level of D-DT is increased in abiological sample obtained from the subject, the level of D-DT iscompared with the level of at least one comparator control contained inthe kit, such as a positive control, a negative control, a historicalcontrol, a historical norm, or the level of another reference moleculein the biological sample. In one embodiment, the reference molecule inthe biological sample is MIF. In certain embodiments, the ratio of D-DTand MIF is determined to aid in the diagnosis.

In another embodiment, the kit comprises the components of a diagnosticassay for diagnosing ischemia-reperfusion injury in a subject in needthereof, containing instructional material and the components fordetermining whether the level of D-DT is reduced in a biological sampleobtained from the subject. In various embodiments, determining whetherthe level of D-DT is reduced in a biological sample obtained from thesubject, the level of D-DT is compared with the level of at least onecomparator control contained in the kit, such as a positive control, anegative control, a historical control, a historical norm, or the levelof another reference molecule in the biological sample. In oneembodiment, the reference molecule in the biological sample is MIF. Incertain embodiments, the ratio of D-DT and MIF is determined to aid inthe diagnosis.

In a further embodiment, the kit comprises the components of an assayfor monitoring the effectiveness of a treatment administered to asubject in need thereof, containing instructional material and thecomponents for determining whether the level of D-DT in a biologicalsample obtained from the subject is modulated during or afteradministration of the treatment. In various embodiments, to determinewhether the level of D-DT is modulated in a biological sample obtainedfrom the subject, the level of D-DT is compared with the level of atleast one comparator control contained in the kit, such as a positivecontrol, a negative control, a historical control, a historical norm, orthe level of another reference molecule in the biological sample. In oneembodiment, the reference molecule in the biological sample is MIF. Incertain embodiments, the ratio of D-DT and MIF is determined to aid inthe monitoring of the treatment.

Therapeutic Inhibitor Compositions and Methods

In various embodiments, the present invention includes D-DT inhibitorcompositions and methods of treating infection, inflammatory disease,autoimmunity and cancer. In various embodiments, the D-DT inhibitorcompositions and methods of treatment of the invention diminish theamount of D-DT polypeptide, the amount of D-DT mRNA, the amount of D-DTenzymatic activity, the amount of D-DT receptor binding activity, or acombination thereof.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that a decrease in the level of D-DTencompasses the decrease in D-DT expression, including transcription,translation, or both. The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, that a decrease inthe level of D-DT includes a decrease in D-DT activity (e.g., enzymaticactivity, receptor binding activity, etc.). Thus, decreasing the levelor activity of D-DT includes, but is not limited to, decreasingtranscription, translation, or both, of a nucleic acid encoding D-DT;and it also includes decreasing any activity of a D-DT polypeptide aswell. The D-DT inhibitor compositions and methods of the invention canselectively inhibit D-DT, or can inhibit both D-DT and MIF.

Inhibition of D-DT can be assessed using a wide variety of methods,including those disclosed herein, as well as methods known in the art orto be developed in the future. That is, the routineer would appreciate,based upon the disclosure provided herein, that decreasing the level oractivity of D-DT can be readily assessed using methods that assess thelevel of a nucleic acid encoding D-DT (e.g., mRNA), the level of a D-DTpolypeptide present in a biological sample, the level of D-DT activity(e.g., enzymatic activity, receptor binding activity), or combinationsthereof.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in treating infection,inflammatory disease, autoimmunity or cancer in a subject in needthereof, whether or not the subject also being treated with othermedication or therapy. Further, the skilled artisan would furtherappreciate, based upon the teachings provided herein, that theinfection, inflammatory disease, autoimmunity or cancer treatable by thecompositions and methods described herein encompass any pathologyassociated infection, inflammatory disease, autoimmunity or cancer whereD-DT plays a role.

The D-DT inhibitor compositions and methods of the invention thatdecrease the level, enzymatic activity, or receptor binding activity ofD-DT include, but should not be construed as being limited to, achemical compound, a protein, a peptide, a peptidomemetic, an antibody,a ribozyme, a small molecule chemical compound, an antisense nucleicacid molecule (e.g., siRNA, miRNA, etc.), or combinations thereof. Oneof skill in the art would readily appreciate, based on the disclosureprovided herein, that a D-DT inhibitor composition encompasses achemical compound that decreases the level or activity of D-DT.Additionally, a D-DT inhibitor composition encompasses a chemicallymodified compound, and derivatives, as is well known to one of skill inthe chemical arts. In a particular embodiment, the D-DT inhibitor is ansiRNA comprising the nucleic acid sequence 5′-GCATGACCCTGTTGATGAA-3′(SEQ ID NO: 2).

The D-DT inhibitor compositions and methods of the invention thatdecrease the level or activity of D-DT include antibodies. Theantibodies of the invention include a variety of forms of antibodiesincluding, for example, polyclonal antibodies, monoclonal antibodies,intracellular antibodies (“intrabodies”), Fv, Fab and F(ab)2, singlechain antibodies (scFv), heavy chain antibodies (such as camelidantibodies), synthetic antibodies, chimeric antibodies, and humanizedantibodies. In one embodiment, the antibody of the invention is anantibody that specifically binds to D-DT, and does not substantiallybind to MIF. In another embodiment, the antibody of the invention is anantibody that specifically binds to D-DT, and also specifically binds toMIF. In a further embodiment, the antibody of the invention is adual-specific antibody that concurrently binds both D-DT and MIF.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that a D-DTinhibitor composition includes such inhibitors as discovered in thefuture, as can be identified by well-known criteria in the art ofpharmacology, such as the physiological results of inhibition of D-DT asdescribed in detail herein and/or as known in the art. Therefore, thepresent invention is not limited in any way to any particular D-DTinhibitor composition as exemplified or disclosed herein; rather, theinvention encompasses those inhibitor compositions that would beunderstood by the routineer to be useful as are known in the art and asare discovered in the future.

Further methods of identifying and producing D-DT inhibitor compositionsare well known to those of ordinary skill in the art, including, but notlimited, obtaining an inhibitor from a naturally occurring source (i.e.,Streptomyces sp., Pseudomonas sp., Stylotella aurantium). Alternatively,a D-DT inhibitor can be synthesized chemically. Further, the routineerwould appreciate, based upon the teachings provided herein, that a D-DTinhibitor composition can be obtained from a recombinant organism.Compositions and methods for chemically synthesizing D-DT inhibitors andfor obtaining them from natural sources are well known in the art andare described in the art.

One of skill in the art will appreciate that an inhibitor can beadministered as a small molecule chemical, a protein, an antibody, anucleic acid construct encoding a protein, an antisense nucleic acid, anucleic acid construct encoding an antisense nucleic acid, orcombinations thereof. Numerous vectors and other compositions andmethods are well known for administering a protein or a nucleic acidconstruct encoding a protein to cells or tissues. Therefore, theinvention includes a method of administering a protein or a nucleic acidencoding a protein that is an inhibitor of D-DT. (Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York; Ausubel et al., 1997, Current Protocols in Molecular Biology,John Wiley & Sons, New York).

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself increases the amount or activity ofD-DT can serve in the compositions and methods of the present inventionto decrease the amount or activity of D-DT.

Antisense oligonucleotides are DNA or RNA molecules that arecomplementary to some portion of an RNA molecule. When present in acell, antisense oligonucleotides hybridize to an existing RNA moleculeand inhibit translation into a gene product. Inhibiting the expressionof a gene using an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931). The methods of the invention include the use of an antisenseoligonucleotide to diminish the amount of D-DT, or to diminish theamount of a molecule that causes an increase in the amount or activityof D-DT, thereby decreasing the amount or activity of D-DT.

Contemplated in the present invention are antisense oligonucleotidesthat are synthesized and provided to the cell by way of methods wellknown to those of ordinary skill in the art. As an example, an antisenseoligonucleotide can be synthesized to be between about 10 and about 100,more preferably between about 15 and about 50 nucleotides long. Thesynthesis of nucleic acid molecules is well known in the art, as is thesynthesis of modified antisense oligonucleotides to improve biologicalactivity in comparison to unmodified antisense oligonucleotides (Tullis,1991, U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing D-DT, or of a geneexpressing a protein that increases the level or activity of D-DT, canbe accomplished through the use of a ribozyme. Using ribozymes forinhibiting gene expression is well known to those of skill in the art(see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al.,1989, Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).Ribozymes are catalytic RNA molecules with the ability to cleave othersingle-stranded RNA molecules. Ribozymes are known to be sequencespecific, and can therefore be modified to recognize a specificnucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowingthe selective cleavage of specific mRNA molecules. Given the nucleotidesequence of the molecule, one of ordinary skill in the art couldsynthesize an antisense oligonucleotide or ribozyme without undueexperimentation, provided with the disclosure and referencesincorporated herein.

One of skill in the art will appreciate that inhibitors of D-DT can beadministered singly or in any combination. Further, D-DT inhibitors canbe administered singly or in any combination in a temporal sense, inthat they may be administered concurrently, or before, and/or after eachother. One of ordinary skill in the art will appreciate, based on thedisclosure provided herein, that D-DT inhibitor compositions can be usedto treat infection, inflammatory disease, autoimmunity or cancer in asubject in need thereof, and that an inhibitor composition can be usedalone or in any combination with another inhibitor to effect atherapeutic result.

In various embodiments, any of the inhibitors of D-DT of the inventiondescribed herein can be administered alone or in combination with otherinhibitors of other molecules associated with infection, inflammatorydisease, autoimmunity or cancer, such as, for example, MIF. In someembodiments, the D-DT inhibitors of the invention selectively inhibitD-DT and do not also inhibit MIF. In other embodiments, the D-DTinhibitors of the invention inhibit D-DT and also inhibit MIF.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder that isalready established. Particularly, the disease or disorder need not havemanifested to the point of detriment to the subject; indeed, the diseaseor disorder need not be detected in a subject before treatment isadministered. That is, significant disease or disorder does not have tooccur before the present invention may provide benefit. Therefore, thepresent invention includes a method for preventing infection,inflammatory disease, autoimmunity or cancer in a subject, in that aD-DT inhibitor composition, as discussed previously elsewhere herein,can be administered to a subject prior to the onset of the infection,inflammatory disease, autoimmunity or cancer, thereby preventing theinfection, inflammatory disease, autoimmunity or cancer. The preventivemethods described herein also include the treatment of a subject that isin remission for the prevention of a recurrence of an infection,inflammatory disease, autoimmunity or cancer.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of infection, inflammatory disease,autoimmunity or cancer encompasses administering to a subject a D-DTinhibitor composition as a preventative measure against infection,inflammatory disease, autoimmunity or cancer. As more fully discussedelsewhere herein, methods of decreasing the level or activity of D-DTencompass a wide plethora of techniques for decreasing not only D-DTactivity, but also for decreasing expression of a nucleic acid encodingD-DT, including either a decrease in transcription, a decrease intranslation, or both.

Additionally, as disclosed elsewhere herein, one skilled in the artwould understand, once armed with the teaching provided herein, that thepresent invention encompasses a method of preventing a wide variety ofdiseases, disorders and pathologies where a decrease in expressionand/or activity of D-DT mediates, treats or prevents the disease,disorder or pathology. Methods for assessing whether a disease relatesto increased levels or activity of D-DT are known in the art. Further,the invention encompasses treatment or prevention of such diseasesdiscovered in the future.

The invention encompasses administration of an inhibitor of D-DT topractice the methods of the invention; the skilled artisan wouldunderstand, based on the disclosure provided herein, how to formulateand administer the appropriate D-DT inhibitor to a subject. Indeed, thesuccessful administration of the D-DT inhibitor has been reduced topractice as exemplified herein. However, the present invention is notlimited to any particular method of administration or treatment regimen.

Therapeutic Activators and Methods

In various embodiments, the present invention includes D-DT activatorcompositions and methods of treating ischemia-reperfusion injury in asubject, a tissue, or an organ in need thereof. In various embodiments,the D-DT activator compositions and methods of treatment of theinvention increase the amount of D-DT polypeptide, the amount of D-DTmRNA, the amount of D-DT enzymatic activity, the amount of D-DT receptorbinding activity, or a combination thereof.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of D-DTencompasses the increase in D-DT expression, including transcription,translation, or both. The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, that an increase inthe level of D-DT includes an increase in D-DT activity (e.g., enzymaticactivity, receptor binding activity, etc.). Thus, increasing the levelor activity of D-DT includes, but is not limited to, increasing theamount of D-DT polypeptide, increasing transcription, translation, orboth, of a nucleic acid encoding D-DT; and it also includes increasingany activity of a D-DT polypeptide as well. The D-DT activatorcompositions and methods of the invention can selectively activate D-DT,or can activate both D-DT and MIF.

Thus, the present invention relates to the prevention and treatment ofischemia-reperfusion injury by administration of a D-DT polypeptide, arecombinant D-DT polypeptide, an active D-DT polypeptide fragment, or anactivator of D-DT expression or activity.

It is understood by one skilled in the art, that an increase in thelevel of D-DT encompasses the increase of D-DT protein expression.Additionally, the skilled artisan would appreciate, that an increase inthe level of D-DT includes an increase in D-DT activity. Thus,increasing the level or activity of D-DT includes, but is not limitedto, increasing transcription, translation, or both, of a nucleic acidencoding D-DT; and it also includes increasing any activity of D-DT aswell.

Activation of D-DT can be assessed using a wide variety of methods,including those disclosed herein, as well as methods well-known in theart or to be developed in the future. That is, the routineer wouldappreciate, based upon the disclosure provided herein, that increasingthe level or activity of D-DT can be readily assessed using methods thatassess the level of a nucleic acid encoding D-DT (e.g., mRNA) and/or thelevel of D-DT polypeptide in a biological sample obtained from asubject.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in ischemia-reperfusion injuryin subjects who, in whole (e.g., systemically) or in part (e.g.,locally, tissue, organ), are being or will be, exposed to less thanadequate oxygen levels. Ischemia-reperfusion injury can occur throughthe loss of ventilation, as well as through the loss of circulation, toall or to part, of the subject's body. In one embodiment, the inventionis useful in treating or preventing ischemia-reperfusion injury in atissue or organ intended for transplantation. The skilled artisan willappreciate, based upon the teachings provided herein, that theischemia-reperfusion injuries treatable by the compositions and methodsdescribed herein encompass any ischemia-reperfusion injury.

A D-DT activator can include, but should not be construed as beinglimited to, a chemical compound, a protein, a peptidomemetic, anantibody, a ribozyme, and an antisense nucleic acid molecule. One ofskill in the art would readily appreciate, based on the disclosureprovided herein, that a D-DT activator encompasses a chemical compoundthat increases the level, enzymatic activity, or receptor bindingactivity of D-DT. In some embodiments, the enzymatic activity istautomerase activity. Additionally, a D-DT activator encompasses achemically modified compound, and derivatives, as is well known to oneof skill in the chemical arts.

In various embodiments, the present invention also includes D-DTactivator compositions and methods of immunostimulation in a subject inneed thereof. Immunostimulation is useful in a variety of settings wherean increase in the immunoactivity is desirable. One such non-limitingexample is the use of a D-DT activator composition to increaseimmunoactivity before, during or after vaccination. Another suchnon-limiting example is the use of a D-DT activator composition toincrease immunoactivity when the subject's immune response is otherwiseinadequate. In various embodiments, the D-DT activator compositions andmethods of immunostimulation of the invention increase the amount ofD-DT polypeptide, the amount of D-DT mRNA, the amount of D-DT enzymaticactivity, the amount of D-DT receptor binding activity, or a combinationthereof.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of D-DTencompasses the increase in D-DT expression, including transcription,translation, or both. The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, that an increase inthe level of D-DT includes an increase in D-DT activity (e.g., enzymaticactivity, receptor binding activity, etc.). Thus, increasing the levelor activity of D-DT includes, but is not limited to, increasing theamount of D-DT polypeptide, increasing transcription, translation, orboth, of a nucleic acid encoding D-DT; and it also includes increasingany activity of a D-DT polypeptide as well. The D-DT activatorcompositions and methods of the invention can selectively activate D-DT,or can activate both D-DT and MIF. Thus, the present invention relatesto immunostimulation by administration of a D-DT polypeptide, arecombinant D-DT polypeptide, an active D-DT polypeptide fragment, or anactivator of D-DT expression or activity.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that a D-DTactivator includes such activators as discovered in the future, as canbe identified by well-known criteria in the art of pharmacology, such asthe physiological results of activation of D-DT as described in detailherein and/or as known in the art. Therefore, the present invention isnot limited in any way to any particular D-DT activator as exemplifiedor disclosed herein; rather, the invention encompasses those activatorsthat would be understood by the routineer to be useful as are known inthe art and as are discovered in the future.

Further methods of identifying and producing a D-DT activator are wellknown to those of ordinary skill in the art, including, but not limited,obtaining an activator from a naturally occurring source (i.e.,Streptomyces sp., Pseudomonas sp., Stylotella aurantium). Alternatively,a D-DT activator can be synthesized chemically. Further, the routineerwould appreciate, based upon the teachings provided herein, that a D-DTactivator can be obtained from a recombinant organism. Compositions andmethods for chemically synthesizing D-DT activators and for obtainingthem from natural sources are well known in the art and are described inthe art.

One of skill in the art will appreciate that an activator can beadministered as a small molecule chemical, a protein, a nucleic acidconstruct encoding a protein, or combinations thereof. Numerous vectorsand other compositions and methods are well known for administering aprotein or a nucleic acid construct encoding a protein to cells ortissues. Therefore, the invention includes a method of administering aprotein or a nucleic acid encoding an protein that is an activator ofD-DT. (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York).

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself diminishes the amount or activity ofD-DT can serve to increase the amount or activity of D-DT. Antisenseoligonucleotides are DNA or RNA molecules that are complementary to someportion of a mRNA molecule. When present in a cell, antisenseoligonucleotides hybridize to an existing mRNA molecule and inhibittranslation into a gene product. Inhibiting the expression of a geneusing an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931). The methods of the invention include the use of antisenseoligonucleotide to diminish the amount of a molecule that causes adecrease in the amount or activity D-DT, thereby increasing the amountor activity of D-DT. Contemplated in the present invention are antisenseoligonucleotides that are synthesized and provided to the cell by way ofmethods well known to those of ordinary skill in the art. As an example,an antisense oligonucleotide can be synthesized to be between about 10and about 100, more preferably between about 15 and about 50 nucleotideslong. The synthesis of nucleic acid molecules is well known in the art,as is the synthesis of modified antisense oligonucleotides to improvebiological activity in comparison to unmodified antisenseoligonucleotides (Tullis, 1991, U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing a protein that diminishesthe level or activity of D-DT can be accomplished through the use of aribozyme. Using ribozymes for inhibiting gene expression is well knownto those of skill in the art (see, e.g., Cech et al., 1992, J. Biol.Chem. 267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA moleculeswith the ability to cleave other single-stranded RNA molecules.Ribozymes are known to be sequence specific, and can therefore bemodified to recognize a specific nucleotide sequence (Cech, 1988, J.Amer. Med. Assn. 260:3030), allowing the selective cleavage of specificmRNA molecules. Given the nucleotide sequence of the molecule, one ofordinary skill in the art could synthesize an antisense oligonucleotideor ribozyme without undue experimentation, provided with the disclosureand references incorporated herein.

One of skill in the art will appreciate that a D-DT polypeptide, arecombinant D-DT polypeptide, or an active D-DT polypeptide fragment canbe administered singly or in any combination thereof. Further, a D-DTpolypeptide, a recombinant D-DT polypeptide, or an active D-DTpolypeptide fragment can be administered singly or in any combinationthereof in a temporal sense, in that they may be administeredsimultaneously, before, and/or after each other. One of ordinary skillin the art will appreciate, based on the disclosure provided herein,that a D-DT polypeptide, a recombinant D-DT polypeptide, or an activeD-DT polypeptide fragment can be used to prevent or treatischemia-reperfusion injury, and that an activator can be used alone orin any combination with another D-DT polypeptide, recombinant D-DTpolypeptide, active D-DT polypeptide fragment, or D-DT activator toeffect a therapeutic result.

One of skill in the art will also appreciate that activators of D-DTgene expression can be administered singly or in any combinationthereof. Further, D-DT activators can be administered singly or in anycombination thereof in a temporal sense, in that they may beadministered simultaneously, before, and/or after each other. One ofordinary skill in the art will appreciate, based on the disclosureprovided herein, that D-DT activators can be used to prevent or treatischemia-reperfusion injury, and that an activator can be used alone orin any combination with another activator, D-DT polypeptide, recombinantD-DT polypeptide, or active D-DT polypeptide fragment to effect atherapeutic result.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of ischemia-reperfusion injuryonce the ischemia-reperfusion injury is established. Particularly, thesymptoms of the ischemia-reperfusion injury need not have manifested tothe point of detriment to the subject; indeed, the ischemia-reperfusioninjury need not be detected in a subject before treatment isadministered. That is, significant pathology from anischemia-reperfusion injury does not have to occur before the presentinvention may provide benefit. Therefore, the present invention, asdescribed more fully herein, includes a method for preventingischemia-reperfusion injury in a subject, in that a D-DT molecule, or aD-DT activator, as discussed elsewhere herein, can be administered to asubject prior to the onset of an ischemia-reperfusion injury, therebypreventing the ischemia-reperfusion injury.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of ischemia-reperfusion injuryencompasses administering to a subject a D-DT polypeptide, a recombinantD-DT polypeptide, an active D-DT polypeptide fragment, or D-DT activatoras a preventative measure against ischemia-reperfusion injury. As morefully discussed elsewhere herein, methods of increasing the level oractivity of a D-DT encompass a wide plethora of techniques forincreasing not only D-DT activity, but also for increasing expression ofa nucleic acid encoding D-DT. Additionally, as disclosed elsewhereherein, one skilled in the art would understand, once armed with theteaching provided herein, that the present invention encompasses amethod of preventing a wide variety of diseases where increasedexpression and/or activity of D-DT mediates, treats or prevents thedisease. Further, the invention encompasses treatment or prevention ofsuch diseases discovered in the future.

The invention encompasses administration of a D-DT polypeptide, arecombinant D-DT polypeptide, an active D-DT polypeptide fragment, or aD-DT activator to practice the methods of the invention; the skilledartisan would understand, based on the disclosure provided herein, howto formulate and administer the appropriate D-DT polypeptide,recombinant D-DT polypeptide, active D-DT polypeptide fragment, or D-DTactivator to a subject. However, the present invention is not limited toany particular method of administration or treatment regimen. This isespecially true where it would be appreciated by one skilled in the art,equipped with the disclosure provided herein, including the reduction topractice using an art-recognized model of ischemia-reperfusion injury,that methods of administering a D-DT polypeptide, a recombinant D-DTpolypeptide, an active D-DT polypeptide fragment, or D-DT activator canbe determined by one of skill in the pharmacological arts.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate D-DT polypeptide,recombinant D-DT polypeptide, active D-DT polypeptide fragment, or D-DTactivator, may be combined and which, following the combination, can beused to administer the appropriate D-DT polypeptide, recombinant D-DTpolypeptide, active D-DT polypeptide fragment, or D-DT activator to asubject.

Pharmaceutical Compositions

Compositions identified as modulators of D-DT can be formulated andadministered to a subject, as now described. For example, compositionsidentified as useful D-DT inhibitors for the treatment and/or preventionof infection, inflammatory disease, autoimmunity or cancer, can beformulated and administered to a subject, as now described. Further,compositions identified as useful D-DT activators, as well as D-DTpolypeptides, recombinant D-DT polypeptides, and active D-DT polypeptidefragments, for the treatment and/or prevention of ischemia-reperfusioninjury can be formulated and administered to a subject, as nowdescribed.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a composition useful for the treatment ofischemia-reperfusion injury, or for the treatment of infection,inflammatory disease, autoimmunity or cancer, disclosed herein as anactive ingredient. Such a pharmaceutical composition may consist of theactive ingredient alone, in a form suitable for administration to asubject, or the pharmaceutical composition may comprise the activeingredient and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these. The activeingredient may be present in the pharmaceutical composition in the formof a physiologically acceptable ester or salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate D-DT modulator thereof,may be combined and which, following the combination, can be used toadminister the appropriate D-DT modulator thereof, to a subject.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between about 0.1 ng/kg/day and 100mg/kg/day.

In various embodiments, the pharmaceutical compositions useful in themethods of the invention may be administered, by way of example,systemically, parenterally, or topically, such as, in oral formulations,inhaled formulations, including solid or aerosol, and by topical orother similar formulations. In addition to the appropriate therapeuticcomposition, such pharmaceutical compositions may containpharmaceutically acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, resealed erythrocytes, andimmunologically based systems may also be used to administer anappropriate modulator thereof, according to the methods of theinvention.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, intravenous, ophthalmic, intrathecal and other known routes ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and U.S. Pat. No. 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent.

Known suspending agents include, but are not limited to, sorbitol syrup,hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gumtragacanth, gum acacia, and cellulose derivatives such as sodiumcarboxymethylcellulose, methylcellulose, andhydroxypropylmethylcellulose. Known dispersing or wetting agentsinclude, but are not limited to, naturally-occurring phosphatides suchas lecithin, condensation products of an alkylene oxide with a fattyacid, with a long chain aliphatic alcohol, with a partial ester derivedfrom a fatty acid and a hexitol, or with a partial ester derived from afatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate,heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, andpolyoxyethylene sorbitan monooleate, respectively). Known emulsifyingagents include, but are not limited to, lecithin and acacia. Knownpreservatives include, but are not limited to, methyl, ethyl, orn-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Knownsweetening agents include, for example, glycerol, propylene glycol,sorbitol, sucrose, and saccharin. Known thickening agents for oilysuspensions include, for example, beeswax, hard paraffin, and cetylalcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, cutaneous, subcutaneous,intraperitoneal, intravenous, intramuscular, intracisternal injection,and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g., sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers.

Such a formulation is administered in the manner in which snuff is takeni.e. by rapid inhalation through the nasal passage from a container ofthe powder held close to the nares. Formulations suitable for nasaladministration may, for example, comprise from about as little as 0.1%(w/w) and as much as 100% (w/w) of the active ingredient, and mayfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, contain 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount fromabout 0.01 mg to 20 about 100 g per kilogram of body weight of theanimal. While the precise dosage administered will vary depending uponany number of factors, including, but not limited to, the type of animaland type of disease state being treated, the age of the animal and theroute of administration. Preferably, the dosage of the compound willvary from about 1 mg to about 100 mg per kilogram of body weight of theanimal. More preferably, the dosage will vary from about 1 μg to about 1g per kilogram of body weight of the animal. The compound can beadministered to an animal as frequently as several times daily, or itcan be administered less frequently, such as once a day, once a week,once every two weeks, once a month, or even less frequently, such asonce every several months or even once a year or less. The frequency ofthe dose will be readily apparent to the skilled artisan and will dependupon any number of factors, such as, but not limited to, the type andseverity of the disease being treated, the type and age of the animal,etc.

Methods of Identifying a D-DT Activators or D-DT Inhibitor

The current invention relates to a methods of identifying a compoundthat modulates the level of D-DT, the enzymatic activity of D-DT, thereceptor binding activity of D-DT, or a combination thereof. In someembodiments, the method of identifying of the invention identifies aD-DT inhibitor compound that decreases the level of D-DT, the enzymaticactivity of D-DT, the receptor binding activity of D-DT, or acombination thereof. In other embodiments, the method of identifying ofthe invention identifies a D-DT activator compound that increases thelevel of D-DT, the enzymatic activity of D-DT, the receptor bindingactivity of D-DT, or a combination thereof.

Other methods, as well as variation of the methods disclosed herein willbe apparent from the description of this invention. In variousembodiments, the test compound concentration in the screening assay canbe fixed or varied. A single test compound, or a plurality of testcompounds, can be tested at one time. Suitable test compounds that maybe used include, but are not limited to, proteins, nucleic acids,antisense nucleic acids, small molecules, antibodies and peptides.

The invention relates to a method for screening test compounds toidentify a modulator compound by its ability to modulate (i.e., increaseor decrease) the level of D-DT, the enzymatic activity of D-DT, thereceptor binding activity of D-DT, or a combination thereof, bymeasuring the level of D-DT, the enzymatic activity of D-DT, thereceptor binding activity of D-DT, or a combination thereof, in thepresence and absence of the test compound.

The test compounds can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al., 1993, Proc. Natl.Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al.,1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladnersupra).

In situations where “high-throughput” modalities are preferred, it istypical to that new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of test compounds potentially havingthe desired activity. Such “combinatorial chemical libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. The compounds thus identified canserve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 The D-Dopachrome Tautomerase (D-DT) Gene Product is a Cytokineand Functional Homolog of Macrophage Migration Inhibitory Factor (MIF)

The functional characterization of human and mouse D-DT (also known asMIF-2) is described herein. Recombinant D-DT was produced and wasdemonstrated to bind with high affinity to CD74, activate the ERK1/2 MAPkinase signaling cascade, and recapitulate many of the inflammatoryfunctions of MIF, including modulation of macrophage migration andglucocorticoidinduced immunosuppression. The administration of ananti-D-DT antibody, like anti-MIF, protects mice from lethal endotoxicshock by reducing the circulating levels of proinflammatory cytokines(TNF-α, IFN-γ, IL-12p70, and IL-1β) and increasing the circulatingconcentration of the anti-inflammatory cytokine, IL-10. An analysis ofclinical samples from patients with sepsis or cancer also revealed thatD-DT is systemically expressed and that circulating levels correlatewith MIF and with the severity of inflammatory disease and malignancy.

Both mouse and human D-DT proteins show conservation in their catalytic,N-terminal proline but they lack MIF's -CXXC- and pseudo(E)LRmotifs, thelatter of which is known to mediate noncognate interactions with CXCR2and CXCR4 (Bernhagen, et al., 2007, Nat Med 13:587-596; Weber, et al.,2008, Proc Natl Acad Sci USA 105: 16278-16283). As described herein, arecombinant, endotoxin-free D-DT protein was produced and it is shownthat D-DT, like MIF, tautomerizes the model substrate,p-hydroxyphenylpyruvate, albeit the measured velocity is ˜10 times lowerthan that analyzed for MIF. D-DT is constitutively expressed indifferent tissues and it is up-regulated by proinflammatory activation.MIF is known to bind to the type II transmembrane protein, CD74, leadingto its intracellular phosphorylation, the recruitment of the coreceptorCD44, and the activation of SRC family kinases and the ERK1/2 MAPKpathway (Shi, et al., 2006 Immunity 25:595-606; Leng, et al., 2003, JExp Med 197:1467-1476). Although the interaction between D-DT and sCD74was reduced in comparison with MIF when measured by both competitionbinding and BIAcore studies, the ERK1/2 activation potential of D-DT andMIF appeared comparable by Western blotting. The KD of sCD74 for D-DTwas 60% of that measured for MIF; D-DT was found to have an ˜3-foldhigher association rate (ka) to sCD74 but to dissociate (kd) ˜11-foldfaster than MIF. Although not wishing to be bound by any particulartheory, these differences in association and dissociation values maydifferentiate the signaling action of the two proteins; e.g., D-DTbinding to the receptor might not always trigger a signaling cascade butmight result in its internalization. MIF showed a steeper dose responsethan D-DT in measurements of macrophage migration inhibition andglucocorticoid overriding, but the resolution of these cell-based assaysmay be too limited to make precise, quantitative conclusions. Theseresults may support the notion that D-DT is a less potent cytokine thatmight be associated with the down-regulation of inflammation.

As described herein, anti-D-DT antibodies were produced that do notcross-react with MIF; these antibodies allowed for measurements of thisprotein in biologic specimens. D-DT was expressed in the serum ofendotoxemic mice at levels that were ˜60% those of MIF. MIF is a high,upstream mediator of septic shock, and immunoneutralization, geneticdeletion, or pharmacologic inhibition of MIF protects from lethal shockinduced by different microbial pathogens, even when administered manyhours after microbial invasion (Bernhagen, et al., 1993, Nature365:756-759; Bozza, et al., 1999, J Exp Med 189:341-346; Calandra, etal., 1998, Proc Natl Acad Sci USA 95:11383-11388; Arjona, et al., 2007,J Clin Invest 117: 3059-3066; McDevitt, et al., 2006, J Exp Med203:1185-1196). Immunoneutralization of D-DT conferred protection fromlethal endotoxemia and this effect was accompanied by a broad reductionin the systemic expression of TNF-α, IL-1β, IL-12p70, and IFN-γ and anincrease in the circulating levels of the anti-inflammatory mediator,IL-10. Given current development of MIF inhibitors for the treatment ofthe inflammatory diseases (Arjona, et al., 2007, J Clin Invest 117:3059-3066; Lolis, et al., 2003, Nat Rev Drug Discov 2:635-645; Cournia,et al. 2009, J Med Chem 52:416-424), it was also of interest todetermine whether the combined neutralization of MIF and D-DT had asynergistic effect. MIF levels are elevated in patients with systemicinflammatory disease or with neoplasia, and in many instances acorrelation between disease severity and MIF levels has been observed(Grieb, et al., 2010, Drug News Perspect 23:257-264). Like MIF, D-DTcirculates at baseline levels in healthy individuals and there is acorrelation in the plasma concentrations of the two mediators.Circulating D-DT levels increase significantly in patients with sepsisor ovarian cancer and show a strong correlation with MIF, consistentwith the explanation that the two mediators may be coordinatelyexpressed in response to similar activating stimuli. The findingsdescribed herein include that D-DT is a close structural and functionalhomolog of MIF, that it binds and activates the MIF receptor, and thattargeting D-DT, similar to MIF, protects mice from lethal endotoxemia.

Like MIF (Bacher, et al., 1997, Am J Pathol 150:235-246), D-DT was foundto be widely expressed in tissues. As described elsewhere herein,macrophages produce 20-fold more MIF than D-DT in response to LPS,consistent with the explanation that there may be cell-specific releaseof these mediators and that in contrast to MIF, nonmacrophage sources ofD-DT contribute more importantly to the systemic expression of D-DT thanMIF. Computational inspection of the D-DT and MIF promoter regions showssimilarities in the presence of serum-responsive, SP-1 and AP-1 bindingelements (alggen.lsi.upc.es). Of note, the human D-DT gene lacks thepolymorphic, CATT5-8 microsatellite repeat that exists in the MIFpromoter (rs5844572) and that regulates MIF expression and is associatedwith the severity of autoimmune inflammatory diseases (Grieb, et al.,2010, Drug News Perspect 23:257-264). Although not wishing to be boundby any particular theory, one difference between MIF and D-DT may lie inthe allele-specific regulation of MIF expression but not D-DT. At theprotein level, D-DT lacks MIF's pseudo(E)LR domain, which is necessaryfor activation of CXCR2. Bernhagen and colleagues have reported that MIFinitiates coordinated receptor interactions between CD74 and CXCR2/CXCR4(Bernhagen, et al., 2007, Nat Med 13:587-596; Weber, et al., 2008, ProcNatl Acad Sci USA 105: 16278-16283). D-DT's high binding affinity toCD74 also may facilitate direct chemotactic effects, perhaps in concertwith MIF or IL-8, which is expressed upon CD74 signaling (Coleman, etal., 2008, J Immunol 181:2330-2337; Binsky, et al., 2007, Proc Natl AcadSci USA 104:13408-13413). D-DT binds the intracellular protein, JAB1,indicating that it might affect the regulation of cell cycle control andsignalosome function, similarly to MIF (Kleemann, et al., 2000, Nature408:211-216). Like MIF, D-DT lacks an N-terminal signal sequence and itmay be produced in sufficient intracytoplasmic concentrations toinfluence these regulatory pathways. In summary, the data disclosedherein identify D-DT to be a cytokine and a close functional homolog ofMIF.

The data described herein support the targeting of D-DT, as well as thesimultaneous targeting of D-DT and MIF, in various diseases and diseasemodels.

The materials and methods employed in this example are now described.

Cloning and Purification of D-DT Protein

The cDNAs for the human and mouse D-DT proteins were prepared byamplification of mRNA from human or murine monocytes and subcloned intothe pET22b expression vector. For native protein expression, a stopcodon was engineered. Bacterial extract was purified using a Q Sepharosecolumn (Amersham) and subsequent HPLC using a C18 column (Amersham).D-DT was refolded using the protocol established for MIF renaturation(Bernhagen, et al., 1994, Biochemistry 33: 14144-14155). Protein puritywas verified by Coomassie and fidelity confirmed by mass spectroscopy.The resulting proteins contained <1 pg LPS/μg protein as quantified bythe PyroGene Recombinant Factor C assay (Cambrex). MIF proteins wereproduced as described earlier (Bernhagen, et al., 1994, Biochemistry 33:14144-14155). D-dopachrome tautomerase activity was assessed using thesubstrate HPP, measuring the change in absorbance at 306 nm for 180 s(Stamps, et al., Biochemistry 39:9671-9678).

Anti-D-DT Antibody and ELISA

Polyclonal antibodies against recombinant mouse or human D-DT wereproduced in rabbits. IgG antibody fractions were isolated by Protein Aagarose affinity chromatography (Pierce) and sterile filtered.Microtiter plates (Nunc) were coated with 15 μg/mL of polyclonalanti-D-DT, washed, and blocked in 1% BSA and 1% sucrose. Samples wereadded and incubated for 2 hours, followed by biotinylated anti-D-DTantibody and a streptavidin-HRP conjugate. The D-DT concentrations werecalculated by extrapolation from a sigmoidal quadratic standard curveusing native D-DT protein (dynamic range, 0-625 pg/mL). For mouse orhuman D-DT, the detection limit was 15 pg/mL.

MIF Receptor Binding Studies

Binding of D-DT to the MIF receptor, CD74, was studied by competitionbinding assay as described previously (Leng, et al., 2003, J Exp Med197:1467-1476). Real-time binding interaction of MIF or D-DT withCD7473-232 was measured by surface plasmon resonance using a BIAcore2000 optical biosensor (Kamir, et al., 2008, J Immunol 180:8250-8261).The MIF receptor sCD74 was immobilized to the chip, and binding of theligands D-DT and MIF was measured in five serial dilutions, three timesfor each dilution sample. Sensorgram response data were analyzed in theBIA evaluation kinetics package and the equilibrium binding constantscalculated in the same experiment.

D-DT Protein Expression in Murine Tissues

Tissues were isolated from C57BL/6 mice, and proteins were analyzed byWestern blot (Bacher, et al., 1997, Am J Pathol 150:235-246). Forimmunohistochemistry, tissue sections from C57BL/6 mice (Mizue, et al.,2005, Proc Natl Acad Sci USA 102:14410-14415) were deparaffinized andantigen retrieval was performed using the Target Retrieval Solution(Dako). The specificity of anti-D-DT antibody staining was establishedby preabsorbing an aliquot of the antibody with a 1,000-fold molarexcess of either D-DT or MIF. Slides were incubated with rabbitanti-D-DT or IgG control antibody (1:50) overnight and visualized withthe Liquid DAB+Substrate Chromogen System (Dako). To allow asemiquantitative comparison of different tissues, all slides weredeveloped for 10 min.

siRNA-Mediated Knockdown of D-DT and MIF

Immortalized murine macrophages (Duewell, et al., 2010, Nature464:1357-1361) were transfected with 50 nM siRNA using HiPerFect(Qiagen). Sequences used were 5′-TCAACTATTACGACATGAA-3′ (SEQ ID NO: 1)for MIF and 5′-GCATGACCCTGTTGATGAA-3′ (SEQ ID NO: 2) for D-DT.

Signal Transduction Studies

Mouse peritoneal macrophages (1×10⁶/well) were rendered quiescent byincubation in 0.1% FBS before stimulation with D-DT or MIF for 2 hours(Mitchell, et al., 1999, J Biol Chem 274:18100-18106). Cells were lysedin RIPA buffer and lysates were run on a 4-12% Bis-Tris NuPage gel(Invitrogen). Immunoblotting was conducted with Abs directed againsttotal ERK1/2, and phospho-ERK-1/2 (Cell Signaling).

Migration and Glucocorticoid Overriding Assays

Migration assays were performed as described previously(Hermanowski-Vosatka, et al., 1999, Biochemistry 38:12841-12849).Briefly, human monocytes were incubated for 20 min with MIF or D-DT.Media with or without 25 ng/mL MCP-1 was added in the lower compartmentof migration chambers and monocytes was added to the transwell (0.5-μmpore size) for 90 min. Cells from the lower migration chamber werelysed, and DNA was fluorescently labeled and enumerated at 480/520 nm.Following the original glucocorticoid overriding methodology of Calandraet al. (Calandra, et al., 1995, Nature 377:68-71), macrophages werepreincubated for 1 hour with 100 nM dexamethasone (Sigma) and MIF orD-DT before adding 100 ng/mL LPS (Sigma). TNF levels in supernatantswere measured by ELISA (eBioscience).

MIF/JAB1 Coimmunoprecipitation

Mif−/−MEFs were lysed in ice-cold buffer and incubated with MIF or D-DT,respectively (Kleemann, et al., 2000, Nature 408:211-216). Twomicrograms of anti-JAB1 (2A10.8; Gene Tex/Biozol) or IgG1 control wasadded and the protein complexes were pulled down with magnetic protein Gbeads (Invitrogen). Blotted proteins were visualized using an anti-MIFor anti-D-DT antibodies and then reprobed with anti-JAB1 antibody.

Endotoxemia Model.

Endotoxemia was induced in female BALB/c mice (8 wk old) by i.p.administration of E. coli LPS 0111:B4 (Sigma) at a dose of 12.5 mg/kgfor serum cytokine measurement and 20 mg/kg for intervention experiments(LD80). For D-DT neutralization studies, mice were injected i.p. with200 μL of rabbit anti-D-DT antiserum or nonimmune serum 2 hours beforeadministration of LPS. Mice were monitored every 4 hours within thefirst 72 hours and then twice daily until death or until 14 days.Cytokine levels were obtained by bleeding mice 4, 24, and 36 hours afterLPS challenge, and serum cytokines were analyzed by Luminex (Bio-Rad).

Patient Samples

Serum concentrations of D-DT and MIF were measured in 85 healthyindividuals and in 37 septic patients hospitalized in the medicalintensive care unit (Lesur, et al., 2010, Crit Care 14:R131). The medianAPACHE II score at the time of intensive care unit admission was 22points (range: 10-36 points). The mortality rate was 27%. The etiologicagents of sepsis were Gram-negative bacteria (43%) and Gram-positivebacteria (49%). Two patients had an infection with Grampositive andGram-negative bacteria and one with fungi. Sera from women withbiopsy-proven ovarian cancer (n=21) were from Yale-New Haven Hospital.

The results of this example are now described.

Purification and Characterization of the D-DT Protein

The genes for MIF and D-DT lie within 0.1 kb of each other in both themouse and human genomes and have a similar organizational relationshipwith nearby genes for matrix metalloproteinase 11 and two theta classGSTs (FIG. 1). The amino acid sequences show 34% identity between humanMIF and human D-DT and 27% identity between murine MIF and murine D-DT.The D-DT proteins share with MIF a canonical N-terminal proline (formedafter posttranslational excision of the initiating methionine), whichcatalyzes substrate tautomerization (Bendrat, et al., 1997, Biochemistry36:15356-15362), but they lack two of the three conserved cysteines(Cys59 and Cys80) that appear in all known mammalian MIF proteins.Murine and human D-DT also lack the pseudo(E)LR (Arg11, Asp44) motifthat mediates MIF's noncanonical interactions with the CXCR2 chemokinereceptor (Weber, et al., 2008, Proc Natl Acad Sci USA 105: 16278-16283).The mRNA for D-DT does not encode either an N-terminal or an internalsecretory signal sequence, suggesting that like MIF, D-DT is secreted bya specialized, nonclassical export pathway (Merk, et al., 2009, JImmunol 182:6896-6906).

The cDNAs for human and mouse D-DT were prepared from monocytes andcloned into a bacterial expression vector for recombinant proteinproduction. Work was performed with native sequence proteins becausestructure-function studies have shown that modifications of the N or Ctermini interfere with trimer formation, the functional unit of MIF(Bendrat, et al., 1997, Biochemistry 36:15356-15362; Sun, et al., 1996,Proc Natl Acad Sci USA 93: 5191-5196; El-Turk, et al., 2008,Biochemistry 47:10740-10756). Recombinant D-DT protein was purified byanion exchange chromatography followed by high performance liquidchromatography (HPLC) (FIG. 2A). Mass spectroscopy of purified mouseD-DT protein gave an m/z of 12,947, which lies within 0.02% of thecalculated mass for D-DT (FIG. 2B). A minor peak of 13,079 Da also wasdetected; this peak corresponds to the molecular mass of D-DT with anuncleaved N-terminal methionine (expected m/z=13,077). MIF tautomerizesmodel substrates such as D-dopachrome and p-hydoxyphenylpyruvate (HPP)(whether a physiological substrate exists is unknown) (Fingerle-Rowson,et al., 2009, Mol Cell Biol 29:1922-1932), and D-DT purified from liverhas tautomerization activity (Rosengren, et al., 1996, Mol Med2:143-149; Odh, et al., 1993, Biochem Biophys Res Commun 197:619-624).It was verified that recombinant D-DT tautomerizes HPP with a specificactivity that is ˜10 times lower than that measured for MIF(D-DT=0.5±0.1 Δ306·min-1·μM-1, and MIF=4.3±1.1 Δ306·min-1·μM-1, P<0.001)(FIG. 2C). A possible explanation for the discrepancy inactivity mightbe structural differences in the active site regions of D-DT and MIF,resulting in a reduced affinity of D-DT to its substrate (Sugimoto, etal., 1999, Biochemistry 38:3268-3279; Sun, et al., 1996, Proc Natl AcadSci USA 93: 5191-5196). A polyclonal anti-D-DT antibody was prepared toestablish an ELISA. This antibody recognized both murine and human D-DTbut did not cross-react with MIF in its denatured form (assessed byWestern blot; FIG. 2D, Left) or in its native form (assessed by ELISA;FIG. 2D, Right).

D-DT Binds the MIF Receptor, CD74, and the Intracellular Protein, JAB1

MIF activates ERK1/2 phosphorylation by engaging CD74, and ahigh-affinity binding interaction between MIF and the CD74 ectodomain(CD7473-232 or sCD74) has been demonstrated by surface plasmon resonance(Leng, et al., 2003, J Exp Med 197:1467-1476). The interaction betweenD-DT and sCD74 in a competition binding assay was studied. D-DT reducedMIF binding to the CD74 ectodomain in a dose-dependent manner, with amaximal effect of ˜50% compared with MIF (FIG. 3A). Measurement of theequilibrium dissociation constants between human D-DT or MIF and sCD74by surface plasmon resonance (BIAcore analysis) revealed a high-affinitybinding interaction between D-DT and the MIF receptor (KD of 5.42×10-9M) (FIG. 3B), albeit 60% lower than for MIF (KD=1.40×10-9 M). Detailedanalysis revealed a ka of 1.2×105 M-1·s-1 for D-DT and only 4.3×104M-1·s-1 for MIF, whereas the dissociation rate (kd) was 11-fold lowerfor MIF than for D-DT (6×10-5·s-1 and 6.6×10-4·s-1, respectively). Thesemeasurements demonstrate that D-DT has an ˜3-fold higher binding rate tothe receptor CD74, but also dissociates much faster than MIF. Theintracellular transcriptional regulator and COPS signalosome componentJAB1 is a well characterized binding partner of MIF that has beenimplicated in MIF-dependent control of cell proliferation (Kleemann, etal., 2000, Nature 408:211-216). D-DT binds to JAB1 as demonstrated bycoimmunoprecipation (FIG. 3C), and the interaction affinity between JAB1and D-DT appears comparable to that observed between JAB1 and MIF.

Differential Expression of D-DT and MIF

The differential expression of D-DT in tissue compared with MIF wasassessed. Esumi et al. have published Northern blotting data for D-DTexpression that suggest enhanced expression in the murine brain comparedwith Mif (Esumi, et al., 1998, Mamm Genome 9:753-757). Eight differentmouse organs were analyzed by Western blotting for D-DT, MIF, and CD74.D-DT and MIF were present in constitutive and appreciable levels in alltissues examined, with the greatest difference observed in the testis,where D-DT appeared in severalfold higher concentrations compared withMIF (FIG. 3D). Immunostaining of murine tissue confirmed these resultsand showed that D-DT, like MIF (Bacher, et al., 1997, Am J Pathol150:235-246), is detected in virtually all cells, with prominentstaining in the epithelia of the kidney, the lung, the bowel,hepatocytes, and the follicular area of the spleen (FIG. 3E).

D-DT Initiates ERK-1/2 Phosphorylation in a MIFReceptor-Complex-Dependent Manner, Mediates Macrophage Migration Arrest,and Counterregulates Glucocorticoid-Induced Immunosuppression

MIF binding to CD74 leads to the recruitment of CD44 and theintracellular phosphorylation of these proteins, resulting in theactivation of SRC family nonreceptor tyrosine kinases and the initiationof the ERK1/2 MAP kinase cascade (Shi, et al., 2006 Immunity 25:595-606;Leng, et al., 2003, J Exp Med 197:1467-1476). Stimulation of culturedmacrophages with increasing concentrations of recombinant D-DT produceda dose-dependent phosphorylation of ERK1/2, with an activating effectthat was both sustained (2 h) (Mitchell, et al., 1999, J Biol Chem274:18100-18106) and comparable to that observed for MIF (FIG. 4A, Top).Costimulation with D-DT and MIF revealed an additive effect of the twoproteins in the ERK1/2 MAP kinase pathway (FIG. 4A, Middle).D-DT-induced ERK1/2 phosphorylation was strictly dependent on theexpression of both CD74 and CD44, as previously reported for MIF (Shi,et al., 2006 Immunity 25:595-606) (FIG. 4A, Bottom).

The biologic activity of recombinant D-DT was analyzed by first assayingfor MIF's effect on macrophage chemotaxis (Hermanowski-Vosatka, et al.,1999, Biochemistry 38:12841-12849). D-DT inhibited chemotaxis induced byCCL2/monocyte chemotactic protein (MCP)-1, but with a less steep doseresponse and reduced inhibitory action at 1 μg/mL compared with MIF(FIG. 4B). Although not wishing to be bound by any particular theory,this observation may be explained by the reduced binding affinity ofD-DT for the MIF receptor or different rates of ligandassociation/dissociation and a consequent reduction in the downstreamevents necessary for CCL2 desensitization. MIF is unique among immunemediators in its ability to counterregulate the immunosuppressive actionof glucocorticoids, which occurs via intracellular pathways that involvecytoplasmic phospholipase A2, IκB1, and MKP-1 (Calandra, et al., 1995,Nature 377:68-71; Flaster, et al, 2007, Mol Endocrinol 21:1267-1280;Roger, et al., 2005, Eur J Immunol 35:3405-3413). Using a standardizedassay (Calandra, et al., 1995, Nature 377:68-71), it was furtherobserved that D-DT, like MIF, counterregulated the inhibitory effect ofglucocorticoids on TNF production from lipopolysaccharide(LPS)-stimulated macrophages (FIG. 4C). Similar to observations in themigration assay, D-DT shows a decreased counterregulatory potential atlow concentrations compared with MIF, which may be attributed to lowerbinding affinity to the MIF receptor.

D-DT is Produced in Response to LPS and Mediates Lethal Endotoxemia

Whereas macrophages have been considered historically to be a maintarget of MIF action, these cells also are a major source of MIFproduction in response to microbial products and tissue invasion in vivo(Calandra, et al, 1994, J Exp Med 179:1895-1902). Cultured macrophageswere stimulated with 1 μg/mL of Escherichia coli LPS and the secretionof D-DT and MIF was quantified by specific ELISA. LPS-stimulatedmacrophages released D-DT into conditioned medium with kinetics thatwere very similar to those of MIF (FIG. 5A). Peak levels were detectableat 16 hours and decreased thereafter. Unstimulated cells also slowlyreleased these proteins into supernatants, which in the case of MIF hasbeen attributed to a low level of constitutive secretion (Merk, et al.,2009, J Immunol 182:6896-6906).

It was next assessed whether there is reciprocal regulation of D-DT andMIF expression in macrophages. D-DT or MIF were depleted in immortalizedmacrophages by siRNA-mediated knockdown but detected no effect on theexpression level of the reciprocal protein in response to LPSstimulation (FIG. 5B). The administration of LPS to mice resulted in atimedependent increase in plasma D-DT concentrations (6.0±4.3 ng/mL to26±12 ng/mL at 24 h), and this increase mimicked the rise observed forMIF (1.0±0.9 ng/mL to 43±28 ng/mL at 24 h) (FIG. 5C). D-DT also isdetectable in plasma under basal conditions and at comparable levels toMIF (D-DT=6.0±4.3 ng/mL and MIF=1.0±0.9 ng/mL) (Calandra, et al., 1995,Nature 377:68-71). It is noteworthy that whereas plasma MIF and D-DTcirculate in similar concentrations under basal or stimulatedconditions, LPS-stimulated macrophages produce 20-fold more MIF thanD-DT (FIG. 5A). Although not wishing to be bound by any particulartheory, this observation is consistent with the explanation thatnonmacrophage sources of D-DT contribute significantly to plasma D-DTexpression in vivo.

Immunoneutralization of D-DT Protects from Lethal Endotoxemia

Immunoneutralization or genetic deletion of MIF protects mice fromendotoxic shock (Bernhagen, et al., 1993, Nature 365:756-759; Bozza, etal., 1999, J Exp Med 189:341-346) and subsequent studies demonstratedthat this protective effect is due to a reduction in the expression oftissue-damaging, effector cytokines such as TNF and IL-1 (Mitchell, etal., 2002, Proc Natl Acad Sci USA 99:345-350; Calandra, et al., 2000,Nat Med 6:164-170). The administration of a specific anti-D-DT antibodybefore the injection of an LD80 dose of LPS increased survival from 20to 79% (FIG. 5D), which is a level of protection comparable to that ofanti-MIF (Bernhagen, et al., 1993, Nature 365:756-759). An analysis ofplasma cytokine expression showed that D-DT neutralization wasassociated with a significant reduction in the circulating concentrationof several proinflammatory cytokines (TNF-α, IL-1β, IFN-γ, and IL-12p70)implicated in shock pathogenesis (FIG. 5E). In contrast, theconcentration of the anti-inflammatory cytokine IL-10 was increased inthe anti-D-DT-treated group compared with controls.

D-DT Expression is Up-Regulated in Patients with Sepsis, InvasiveCancer, or Vasculitis

To determine whether D-DT is up-regulated in human disease and to assesspotential relationships between D-DT and MIF production in vivo, theserum concentrations of these mediators were analyzed in individualswith sepsis or with ovarian cancer, which are two conditionscharacterized by high levels of MIF in plasma (Calandra, et al., 2000,Nat Med 6:164-170; Visintin, et al., 2008, Clin Cancer Res14:1065-1072). There was a statistically significant increase incirculating D-DT protein in patients with sepsis compared with healthycontrols (sepsis patients, 55.5±61.3 ng/mL; control group, 5.9±3.9ng/mL; P<0.0001) (FIG. 6A). MIF levels also were elevated, as expectedfrom prior work (Calandra, et al., 2000, Nat Med 6:164-170; Bozza, etal., 2004, Shock 22:309-313; Lehmann, et al., 2001, Intensive Care Med27:1412-1415) (sepsis patients, 111.0±69.0 ng/mL; control group, 6.3±6.2ng/mL; P<0.0001). Receiver operator characteristic (ROC) analysisrevealed an area under the curve of 0.99 for MIF or D-DT, indicatingthat both proteins show excellent sensitivity and specificity for thediagnosis of sepsis. These measurements further revealed that serumlevels of D-DT, like MIF (Bozza, et al., 2004, Shock 22:309-313; Emonts,et al., 2007, Clin Infect Dis 44:1321-1328), correlate with diseaseseverity as determined by APACHE II clinical severity scores (FIG. 6B).Both D-DT and MIF show a significant association with outcomeparameters; however, a more precise assessment of the prognostic valueof these proteins may be obtained by serial measurements. Serum D-DT andMIF concentrations also correlate in healthy individuals, and thecorrelation coefficient increases from R=0.32 to R=0.5 for the analysisof these mediators in patients with sepsis (FIG. 6C). Circulating D-DTconcentrations were also found to be elevated in patients with ovariancancer (cancer patients, 15.2±13.8 ng/mL; control group, 5.9±3.9 ng/mL;P<0.0001) (FIG. 6D). ROC analysis revealed an area under the curve of0.8, which is comparable to that observed for MIF (0.7). The correlationbetween MIF and D-DT serum concentrations was stronger and showedgreater statistical significance than that observed for septic patients(R=0.9, P=0.0001). D-DT expression is up-regulated in subjects withvasculitis and D-DT concentrations correlate with MIF and the presenceof vasculitis (FIG. 7).

Example 2 Ischemia-Reperfusion Injury

As described herein, D-DT activates the cardioprotective AMPK pathway inrat heart muscles. Mouse recombinant DDT activates AMP-activated proteinkinase in rat heart left ventricular papillary muscles. DDT incubationleads to phosphorylation of threonine 172, the major activating site inthe catalytic alpha subunit of AMPK, as well as downstream targetacetyl-CoA carboxylase (ACC). AMPK pathway activation by DDT is dose andtime dependent (FIGS. 8 and 9).

Moreover, endogenous DDT has a role in mediating AMPK activation duringhypoxia in isolated rat heart left ventricular muscles (FIG. 9). Theaddition of purified rabbit polyclonal neutralizing D-DT antibody for 30minutes prior to and during 15 minutes of hypoxia significantly reducedthe critical phosphorylation of threonine 172 in the activating domainof the alpha catalytic subunit of AMP-activated protein kinase. DDTantibody also decreased the phosphorylation of downstream acetyl-CoAcarboxylase. Control was performed with non-immune IgG incubation. FIG.9B shows a comparison to incubation with MIF neutralizing antibody.p=0.05.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of diagnosing a disease or disorder in a subject in needthereof, the method comprising: a. determining the level of D-DT in abiological sample from the subject, b. comparing the level of D-DT inthe biological sample with a comparator control, and diagnosing thesubject with a disease or disorder when the level of D-DT in thebiological sample is different than the level of D-DT of the comparatorcontrol.
 2. The method of claim 1, wherein the level of D-DT in thebiological sample is elevated when compared with the comparator control.3. The method of claim 1, wherein the level of D-DT in the biologicalsample is reduced when compared with the comparator control.
 4. Themethod of claim 1, wherein the level of D-DT in the biological sample isdetermined by measuring the level of D-DT mRNA in the biological sample.5. The method of claim 1, wherein the level of D-DT in the biologicalsample is determined by measuring the level of D-DT polypeptide in thebiological sample.
 6. The method of claim 1, wherein the level of D-DTin the biological sample is determined by measuring an enzymaticactivity of D-DT polypeptide in the biological sample.
 7. The method ofclaim 1, wherein the level of D-DT in the biological sample isdetermined by measuring the binding of a detectable molecule to the D-DTenzyme substrate binding site.
 8. The method of claim 1, wherein thelevel of D-DT in the biological sample is determined by measuring thedisplacement of a detectable molecule from the D-DT enzyme substratebinding site.
 9. The method of claim 1, wherein the comparator controlis at least one selected from the group consisting of: a positivecontrol, a negative control, a historical control, a historical norm, orthe level of a reference molecule in the biological sample. 10.(canceled)
 11. The method of claim 2, wherein the disease or disorder isat least one selected from the group consisting of: infection,inflammatory disease, autoimmunity and cancer.
 12. The method of claim3, wherein the disease or disorder is an ischemia-reperfusion injury.13. The method of claim 1, further comprising the step of treating thesubject for the diagnosed disease or disorder.
 14. A compositioncomprising a D-DT inhibitor.
 15. The composition of claim 14, whereinthe D-DT inhibitor is an antibody that specifically binds to D-DT. 16.The composition of claim 15, wherein the antibody specifically binds toD-DT and does not specifically bind to MIF.
 17. The composition of claim15, wherein the antibody specifically binds to D-DT and alsospecifically binds to MIF.
 18. The composition of claim 15, wherein theantibody is at least one selected from the group consisting of: apolyclonal antibody, a monoclonal antibody, an intracellular antibody,an antibody fragment, a single chain antibody (scFv), a heavy chainantibody, a synthetic antibody, a chimeric antibody, and humanizedantibody.
 19. The composition of claim 14, wherein the D-DT inhibitor isan antisense nucleic acid.
 20. The composition of claim 19, wherein theantisense nucleic acid is at least one selected from the groupconsisting of: siRNA and miRNA.
 21. The composition of claim 20, whereinthe siRNA comprises the nucleic acid sequence of SEQ ID NO:
 2. 22. Thecomposition of claim 14 wherein the D-DT inhibitor is at least oneselected from the group consisting of: a chemical compound, a protein, apeptide, a peptidomemetic, a ribozyme, and a small molecule chemicalcompound.
 23. A method of treating or preventing a disease or disorderin a subject in need thereof, the method comprising: administering tothe subject a therapeutically effective amount of a compositioncomprising a D-DT inhibitor.
 24. The method of claim 23, wherein theD-DT inhibitor is an antibody that specifically binds to D-DT.
 25. Themethod of claim 24, wherein the antibody specifically binds to D-DT anddoes not specifically bind to MIF.
 26. The method of claim 24, whereinthe antibody specifically binds to D-DT and also specifically binds toMIF.
 27. The method of claim 24, wherein the antibody is at least oneselected from the group consisting of: a polyclonal antibody, amonoclonal antibody, an intracellular antibodies, an antibody fragment,a single chain antibody (scFv), a heavy chain antibody, a syntheticantibody, a chimeric antibody, and humanized antibody.
 28. The method ofclaim 23, wherein the D-DT inhibitor is an antisense nucleic acid. 29.The method of claim 28, wherein the antisense nucleic acid is at leastone selected from the group consisting of: siRNA and miRNA.
 30. Themethod of claim 29, wherein the siRNA comprises the nucleic acidsequence of SEQ ID NO:
 2. 31. The method of claim 23, wherein the D-DTinhibitor is at least one selected from the group consisting of: achemical compound, a protein, a peptide, a peptidomemetic, a ribozyme,and a small molecule chemical compound.
 32. The method of claim 23,wherein the disease or disorder is at least one selected from the groupconsisting of: infection, inflammatory disease, autoimmunity and cancer.33-44. (canceled)
 45. A composition comprising a D-DT activator.
 46. Thecomposition of claim 45, wherein the D-DT activator is at least oneselected from the group consisting of: a chemical compound, a protein, apeptide, a peptidomemetic, an antisense nucleic acid, a ribozyme, or asmall molecule chemical compound. 47-63. (canceled)